Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease

ABSTRACT

Provided are tricyclo-DNA (tc-DNA) AON and methods employing tc-DNA AON for modifying splicing events that occur during pre-mRNA processing. Tricyclo-DNA (tc-DNA) AON are described that may be used to facilitate exon skipping or to mask intronic silencer sequences and/or terminal stem-loop sequences during pre-mRNA processing and to target RNase-mediated destruction of processed mRNA. Tc-DNA AON described herein may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping a mutated exon 23 or exon 51 within a dystrophin gene to restore functionality of a dystrophin protein; in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein; and in methods for the treatment of Steinert&#39;s Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats.

BACKGROUND OF THE DISCLOSURE

1. Technical Field

The present disclosure relates, generally, to synthetic antisenseoligonucleotides (AON) and methods employing antisense oligonucleotidesfor modifying splicing events that occur during pre-mRNA processing orfor down-regulating the expression of mutated mRNA that contain repeatedsequences such as, for example, 3′ or 5′ CUG, CAG, and/or CCUG. Morespecifically, disclosed herein are tricyclo-DNA (tc-DNA) AON that areeffective in facilitating exon skipping during pre-mRNA processing, inmasking intronic silencer sequences and/or stem-loop sequences inpre-mRNA, and in targeting the RNase-mediated destruction of mRNA.Described herein are tc-DNA AON that may be used in methods for thetreatment of Duchenne Muscular Dystrophy by skipping mutated exons, suchas a mutated exon 23 or exon 51, within a dystrophin gene to restorefunctionality of a dystrophin protein. Also described are tc-DNA AONthat may be used in methods for the treatment of Spinal Muscular Atrophyby masking an intronic silencing sequence and/or a terminal stem-loopsequence within an SMN2 gene to yield modified functional SMN2 protein,including an amino acid sequence encoded by exon 7, which is capable ofat least partially complementing a non-functional SMN1 protein. Stillfurther tc-DNA AON described herein may be used in methods for thetreatment of Steinert's Myotonic Dystrophy by targeting the destructionof a mutated DM1 mRNA comprising 3′-terminal CUG repeats. Thus, tc-DNAAON and one or more of the foregoing approaches can be used to restorefunctionality in a protein involved in a myopathy.

2. Description of the Related Art

Duchenne Muscular Dystrophy (DMD) is the most common hereditarymyopathy, afflicting about one in 3,500 males regardless of ethnicity.Although infrequent, girls and women may present Duchenne-like symptomsin manifesting carriers. The foremost consequence of DMD is that musclefibers become particularly fragile and natural muscle activity provokesgeneral damage in muscle tissue. The end-point observed in DMD, as wellas in many muscle dystrophies, is that slow degeneration leads to almostcomplete fibrosis with fatty infiltration. Because of spine deformationand breathing difficulties, life expectancy in the 1960s was about 15years. In the absence of cardiac complications, modern improvements inmanagement methods (i.e. arthrodesis and tracheotomy ventilation) haveincreased life expectancy to 30 years.

Clinical symptoms of DMD are evident at the age of 18 months to threeyears and include a delayed ability to walk and climb, difficultygetting up from the floor, and abnormally enlarged calves. At about 5 to6 years, muscle contractions develop in the foot, knee, and hip joints.Progression of the disease is characterized by a continual musclewasting, leading at about 9 to 12 years to the loss of walking ability.In addition, some Duchenne boys present mental retardation suggestingthat the missing protein is also involved in the central nervous system.

Duchenne Muscular Dystrophy is an X-linked recessive disorder. The DMDlocus was identified on the X-chromosome (Xp21.2-OMIMid: 310200) in1986, through a positional cloning approach, in a gene that encodes aprotein called dystrophin. Mutations in the dystrophin gene result in afailure to produce dystrophin in striated muscles. Mothers of affectedboys have a two-thirds chance of carrying a dystrophin mutation, whileapproximately one-third of patients have de novo mutations. More thanhalf of DMD boys exhibit large genomic deletions encompassing one toseveral exons; few of them have large sequence duplications. Others havepoint mutations or very small deletions or duplications that aredifficult to identify.

The extent of the mutations does not, however, directly correlate withthe severity of the phenotype. Out-of-frame deletions or non-sensemutations that yield premature stop codons and subsequent abortion oftranslation result in dystrophin deficiencies characterized by severephenotypes. In-frame deletions are responsible for a milder myopathyknown as Becker muscular dystrophy (BMD).

With nearly 2.5 million base pairs, the DMD locus is the longest geneever detected, but only about 14,000 base pairs contain codingsequences, which are spread over 79 exons. Full length dystrophin (DP427) is a 427 kDa cytoskeletal protein expressed in all muscles, but avariety of protein isoforms (DP 260, DP 140, DP 116, DP 71) aregenerated by the tissue-specific, differential usage (in the retina,central nervous system, peripheral nervous system, and non-muscletissues) of four internal promoters located in introns 29, 43, 55, and62, respectively.

Full-length dystrophin is an essential component of a sarcolemmalglycoprotein complex (SGC) involved in sustaining the membrane integrityof muscle fibers by linking myofiber cytoskeleton to the extracellularmatrix. Sequence analysis has predicted that the dystrophin proteinentails several domains and repeats. Schematically, there is anactin-hybridizing site at the N-terminus (N-ABD); a central rod domain(RD; having 24 spectrin-like repeats) containing four hinge segments (H)that may confer flexibility; and a cystein-rich domain (CRD), whichbinds other members of the DPC, near the C-terminus (CT).

Structure/function analysis has identified domains which are crucial forprotein function. This was exemplified by internal deletions occurringin some patients with a mild disease in whom the deletion encompassedexons 17 to 48 (46% of the coding sequence). England et al., Nature343(6254):180-2 (1990). This led to the concept of functional“minidystrophin” extensively used in the past 10 years in gene transferexperiments. It is now established that removal of the N-ABD and CTdomains cause moderate loss of function, while the CRD is essential.Alterations of the RD result in diverse phenotypes depending on theextent and nature of the truncation. As an example, an RD deleteddystrophin (ΔR1-R24) is not functional, whereas a (ΔH2-R19) truncateddystrophin, which retains eight complete spectrin-like repeats out of24, results in a protein with full activity.

There are two well-characterized genetic animal models for DuchenneMuscular Dystrophy. The mdx mouse harbors a non-sense mutation in exon23 of the dystrophin gene, which precludes the synthesis of full-length,wild-type dystrophin protein. The mdx mouse displays a compensatorymechanism counteracting the degeneration, which could maintain theregeneration process to restore the mechanical damage. The mdx mousedoes not exhibit symptoms of DMD and its life span is almost normal.

The GRMD (Golden Retriever Muscle Dystrophy) dog lacks functionaldystrophin because of a splice site mutation in intron 6, which disruptsthe reading frame. In GRMD, as with human DMD, the progressivedegradation of fibers leads inexorably to skeletal musculature wastingwith marked endomysial and perimysial fibrosis. Because of its DMD-likephenotype, GRMD remains the best available model for the evaluation ofpotential therapies for DMD.

Despite the identification and characterization of mutations in thedystrophin gene that are associated with an onset of DMD and theavailability of suitable animal model systems for testing prospectivetherapeutic agents, there remains a need in the art for compositions andmethods for the treatment of this disease. Several studies over the past10 years support the benefit of steroid treatment (prednisone anddeflazacort) in Duchenne boys, although a broad statistical evaluationhas not yet been fully completed. Pharmacologic-induced read-through ofpremature stop-codon mutations by means of gentamicin medication couldalso potentially be effective in up to 5% of patients with DMD. Clinicaltrials are being carried out in the United States and Italy, even thoughthe results of preclinical studies in the mdx mouse model werecontroversial. A new drug (PTC124) developed by PTC Therapeutics seemsmore promising. Studies are also underway to upregulate the utrophingene using drugs whose product, the dystrophin-like protein utrophin,can compensate for the function of the missing dystrophin.

There are many other avenues of research; as an example, it has beenrecently shown that antagonizing myostatin by using blocking antibodiescould improve muscle strength in mdx mice. This approach was initiallybased on multiple injections of normal myoblasts into the diseasedmuscles. Partridge et al., Nature 337(6203):176-9 (1989). Subsequentclinical trials (1991-98) have failed, although improving cellmanufacturing and delivery procedures have made possible a new phase Itrial in Canada (2002). Recent developments have also provided evidencethat stem cells from either bone marrow or vascular origins can targetskeletal muscle through the systemic pathway, even though the extent ofthe genetic correction is still insufficient.

Gene therapy for DMD lies on in situ delivery of dystrophin mini-genesinto skeletal fibers by using gene vectors as vehicles. A firstexploratory study using naked full length cDNA in a plasmid vector wascarried out in France (2000-03). Among the different types of vectorsthat have been tested for muscle gene therapy, adenovirus associatedvirus (AAV)-derived vectors seem to be the most promising. AAV vectorshave a number of advantages: (i) they are able to infect a wide varietyof cell types including muscle fibers; (ii) they appear safe becausethey lack all viral genes and that wild type viruses have not yet beenassociated with any pathology in human; (iii) conversely to wild typeAAVs, which integrate into the genome of the host cells, replicationdeficient AAV vectors generally persist as episomes thus limiting therisk of insertional mutagenesis or activation of oncogenes; and (iv) incontrast to other vector systems, AAV vectors do not trigger asignificant immune response thus granting long term expression of thetherapeutic transgenes (provided their gene products were not rejected).AAV vectors can also be produced at high titer and forced intra-arterialinjections make them able to achieve gene transfer to significant muscleterritories through a single injection, at least in rodents. AlthoughAAV vectors lack all viral genes, their cargo shipment is limited to 4.5kb. For that reason, the choice of AAV led to the development ofμ-dystrophin variants of about 4 kb instead of the full-lengthdystrophin (14 kb). Several of these variants have been beneficiallytested in the mdx model by either transgenesis or gene transfer.

In many DMD patients as well as in the mdx mouse and the GRMD dog, raredystrophin-positive fibers have been reported. Although the proportionof revertant fibers increases with time, their number is unfortunatelytoo low to confer a significant clinical benefit. The mechanisminitiating these revertant fibers remains unknown although studiessuggest that the reading-frame may be restored by exon-skipping. Such anatural phenomenon has prompted investigation into the design ofstrategies for gene repair/modulation based on the use of 2′-O-methylantisense oligoribonucleotides as well as Morpholinos to interfere withsplicing, thus inducing exon skipping. Indeed, this approach has beensuccessfully used in vitro in mdx, GRMD and DMD muscle cells as well asin vivo (successful phase 1 clinical trial for 2′-O-methyl inNetherlands; a phase 1 with Morpholinos is ongoing in UK). Nevertheless,the weakness of this approach is that it requires regular administrationof the synthetic AOs, and systemic delivery has not been fully achieved.

An alternative approach is to synthesize the sequences of interest insitu from vectors as antisense RNA molecules. Even so, producing“therapeutic” antisense RNA molecules in vivo poses many problems suchas stability and subcellular localization. Small nuclear RNAs (snRNAs),which are known to participate in the splicing reaction, may be used ascarriers to overcome these limitations. Recent reports have shown thatU7 snRNA carrying antisense sequences against the splice junctions ofeither exon 23 or exon 51 of the dystrophin gene induce dystrophinsynthesis in vitro as well as in vivo in mdx and Δ48-50 DMD cells,respectively.

An in silico search of all DMD patients with an out-of-frame deletionwho would theoretically benefit from the skipping of a single exonadjacent to the deletion (on either side) has been performed.Interestingly, it is predicted that skipping exon 51 should restore amini-dystrophin in 22% of the cases (i.e. Δ45-50, Δ47-50, Δ48-50,Δ49-50, Δ50 and Δ52). The resulting truncated proteins are expected tobe at least partially functional since they correspond to deletions thathave been found in some BMD patients. Additionally, a few healthy malescarrying Δ51-52 and Δ48-51 in-frame deletions have been identified.Skipping of exon 51, in select patients, should bring about theproduction of a functional shorter dystrophin thus improving thephenotype.

Mental retardation is a symptom frequently associated with DMD and canresult from the lack of dystrophin in neuronal cells. Rescuing a semifunctional dystrophin in the brain could therefore correct or improvethe cognitive impairment.

Spinal Muscular Atrophy (SMA) refers, generally, to a variety ofdisorders deriving from a common genetic defect in a survival motorneuron (SMN) gene, which, in 1990, was mapped to chromosome 5q11.2-13.3.Human chromosome 5 contains a large duplication such that there are twocopies of the SMN gene, SMN1 and SMN2.

SMA is the most common cause of genetically determined neonatal death.All forms of SMN-associated SMA have a combined incidence of about 1 in6,000. The gene frequency is around 1:80 and approximately one in 40persons is a carrier. There are no known health consequences of being acarrier and the only way one may know to consider the possibility is ifa relative is affected.

SMA is characterized by the loss of the motor neurons of the spinal cordand brainstem. In general, the earlier the symptoms appear, the shorterthe expected life-span. Once symptoms appear, the motor neuron cellsquickly deteriorate. All forms of SMA have in common weakness caused bydenervation, that is, the muscle atrophies because it has lost thesignal to contract due to loss of the innervating nerve. Spinal muscularatrophy only affects motor nerves. Heritable disorders that cause bothweakness due to motor denervation along with sensory impairment due tosensory denervation are known by the inclusive label Charcot-Marie-Toothor Hereditary Motor Sensory Neuropathy.

The course of SMA is directly related to the severity of weakness.Infants with the severe form of SMA frequently succumb to respiratorydisease due to weakness of the muscles that support breathing. Childrenwith milder forms of SMA naturally live much longer although they mayneed extensive medical support, especially those at the more severe endof the spectrum.

Type I SMA, also known as severe infantile SMA or Werdnig Hoffmanndisease, is the most severe, and manifests in the first year of life.This type generally onsets quickly and unexpectedly after birth; babiesdiagnosed with Type I SMA do not generally live past one year of age.Pneumonia is considered the ultimate cause of death due to deteriorationof survival motor neurons; motor neuron death causes insufficientfunctioning of the major bodily organ systems, particularly respiratory(e.g., breathing and ridding of pooled secretions inside lungs). Type IISMA, or intermediate SMA, describes those children who are never able tostand and walk, but who are able to maintain a sitting position at leastsome time in their life. The onset of weakness is usually recognizedsome time between 6 and 18 months. Weakness slowly and graduallyincreases over the life of the individual. Type III SMA patients areable to walk at some time.

SMA is typically diagnosed with a survival motor neuron (SMN) gene test,which determines whether there is at least one copy of a functional SMN1gene, which is distinguished from the highly similar SMN2 gene, by thepresence of exons 7 and 8 in fully-processed mRNA. The SMN2 gene alsocontains a mutation that makes it less efficient at making protein,though it does so in a low level. SMA is caused by loss of the SMN1 genefrom both chromosomes and the inability of SMN2 protein to compensatefor the loss in functional SMN1 protein.

Current strategies for developing SMA therapeutics include identifyingdrugs that increase SMN2 levels, enhance residual SMN2 function, orotherwise compensate for the loss of SMN1 activity. Drugs such asbutyrates, valproic acid, hydroxyurea, and riluzole (Rilutek®, SanofiAventis) are or have been under clinical investigation for the treatmentof SMA. Although gene replacement strategies are being tested inanimals, current treatment for SMA consists of prevention and managementof the secondary effect of chronic motor unit loss. There is currentlyno drug known to alter the course of SMA and it is likely that genereplacement for SMA will require many more years of investigation beforeit can be applied to humans.

Myotonic Dystrophy (DM) is a chronic, slowly progressing, highlyvariable inherited multisystemic disease that can manifest at any agefrom birth to old age. Myotonic dystrophy is the most common form ofadult onset muscular dystrophy and the second most common form of anyskeletal muscle disease after Duchenne muscular dystrophy. DM ischaracterized by wasting of the muscles (muscular dystrophy), posteriorsubcapsular iridescent cataracts (opacity of the lens of the eyes),heart conduction defects, endocrine changes and myotonia (difficultyrelaxing a muscle).

There are currently two known types of adult onset DM, both identifiableby DNA analysis: Myotonic dystrophy type 1 (DM1) is commonly referred toas Steinert's disease, which has a congenital form that can severelyaffect babies and a childhood onset form. Myotonic dystrophy type 2(DM2) is known as PROMM or proximal myotonic myopathy. Additional formsof myotonic dystrophy (e.g., DM3, DM4, DMX) are suspected, but theirexistence remains unproven. While both DM1 and DM2 are considered to beslowly degenerative conditions, DM2 is considered to be generally milderthan DM1.

Presentation of symptoms varies considerably by form (DM1/DM2), severityand even unusual DM2 phenotypes. DM1 patients often present withmyotonia, disabling distal weakness and severe cognitive problems. DM2patients commonly present with muscle pain, stiffness, fatigue, or thedevelopment of proximal lower extremity weakness. Day et al. Neurology60(4): 657-64 (2003). The characteristic pattern of weakness isdifferent for DM1 and DM2. In DM1, it is noted in face and jaw muscles,the drooping of the eyelids (ptosis), weakness of the neck muscles,hands and lower legs. In DM2, the weakness is more evident in proximalmuscles, those closer to the trunk of the body, neck, shoulders, hipflexors and upper legs.

DM1 symptoms include hypersomnia (daytime sleepiness), muscle wasting,dysphagia, and respiratory insufficiency. DM1 patients may experience amore diverse range of cognitive problems than DM2 patients. Depending onwhat form they have and the degree of severity, DM1 cognitive problemsmay range from developmental delays, learning problems, language,speech, behavior, apathy, or hypersomnia. Cognitive manifestations forDM2 include problems with executive function (i.e. organization,concentration, word-finding etc.) and hypersomnia.

In DM1, the affected gene is called DMPK (myotonic dystrophy proteinkinase) and codes for a serine/threonine protein kinase expressed inskeletal muscle. The gene is located on the long arm of chromosome 19.In DM1, the DMPK gene is characterized by a triplet repeat ofCytosine-Thymine-Guanine (CTG). The number of repeats varies greatlyfrom person to person, but the average number in a healthy person isbetween 5 and 37. Sometimes when repetitive sequences of DNA arerepaired or replicated during cell division, the cellular machineryslips and an extra copy of the triplet repeat is added to the sequence.Once there are more than 37 triplet repeats in the DMPK gene thesequence becomes unstable and slippage becomes more common.

People affected with DM1 have over 50 and can have as many as 2000 CTGrepeats. The result being that the repeat size of an individual with DM1will become larger usually during gametogenesis or early embryonicdevelopment, such that children of an affected adult typically exhibitlarger expansions than their parent due to slippage during gametogenesis(this phenomenon is referred to as anticipation). Individuals withlarger expansions have an earlier onset of the disorder and a moresevere phenotype.

DM2 is similarly caused by a defect of the ZNF9 gene on chromosome 3q21.The repeat expansion for DM2 is much larger than for DM1, ranging from75 to over 11,000 repeats and involves a repeat of four nucleotides.Unlike DM1, however, the size of the repeated DNA expansion does notappear to make a difference in the age of onset or disease severity inDM2. Anticipation appears to be less significant in DM2.

There is currently no cure for or treatment specific to myotonicdystrophy. Heart problems, cataracts, and other abnormalities associatedwith the condition can be treated but not cured. There are, however,medical interventions and medications that may relieve some of thesymptoms such as myotonia, pain, and excessive sleepiness. Research inareas such as high throughput screening and antisense therapy hold hopefor more effective targeted treatments for the future. Altered splicingof the muscle-specific chloride channel 1 (ClC-1) causes the myotonicphenotype of DM1 and is reversible in mouse models using Morpholinoantisense oligonucleotides that modify the splicing of ClC-1 mRNA.Wheeler et al., J. Clin. Invest. 117(12):3952-7 (2007).

Despite the ongoing search for therapeutic modalities for DuchenneMuscular Dystrophy, Spinal Muscular Atrophy, and Steinert's MyotonicDystrophy, there remains an urgent need for efficacious compounds andtherapeutic methods for the treatment of these diseases.

SUMMARY OF THE DISCLOSURE

The present disclosure fulfills these and other related needs byproviding tricyclo-DNA (tc-DNA) antisense oligonucleotides (AON) andmethods employing tc-DNA AON for the treatment of diseases such asDuchenne Muscular Dystrophy, Spinal Muscular Atrophy, and Steinert'sMyotonic Dystrophy.

The invention also relates, generally, to a method of correctingabnormal gene expression in a cell of the central nervous system of asubject, the method comprising administering to the subject a tc-DNAantisense oligonucleotide, wherein said tc-DNA antisense oligonucleotideis complementary to a portion of an RNA encoded by said gene, andwherein said tc-DNA antisense oligonucleotide is administeredperipherally to the subject in an amount sufficient to correct saidabnormal expression.

The invention also relates to a method of treating a genetic diseasecaused by abnormal gene expression in the central nervous system of asubject, the method comprising administering to the subject a tc-DNAantisense oligonucleotide, wherein said tc-DNA antisense oligonucleotideis complementary to a portion of an RNA encoded by said gene, andwherein said tc-DNA antisense oligonucleotide is administeredperipherally to the subject in an amount effective to correct saidabnormal expression.

The invention also relates to a pharmaceutical composition comprising atc-DNA antisense oligonucleotide wherein said tc-DNA antisenseoligonucleotide is complementary to a portion of an RNA encoded by ahuman gene, and wherein said composition further comprises apharmaceutical acceptable excipient.

The invention also relates to a tc-DNA antisense oligonucleotide for usein the treatment of a genetic disease caused by abnormal gene expressionin the central nervous system of a subject, said tc-DNA antisenseoligonucleotide being complementary to a portion of an RNA encoded bysaid gene, and said tc-DNA antisense oligonucleotide being administeredperipherally to the subject in an amount effective to correct saidabnormal expression.

As used herein, the term “peripheral administration” includes, withoutlimitation, any administration route which does not imply directinjection into the central nervous system of the subject in need of thetreatment. More particularly, peripheral administration comprisessystemic injections, such as intramuscular (i.m.), intravenous (i.v.),intraperitoneal (i.p.), intra-arterial, sub-cutaneous or transdermicinjections.

The invention also relates to a tc-DNA antisense oligonucleotide for usein the treatment of a neuromuscular or musculoskeletal disease. Theimplemented tc-DNA antisense oligonucleotide is as herein described infurther details below. More particularly, the tc-DNA antisenseoligonucleotide may be one of the specific tc-DNA presented herein.

The neuromuscular or musculoskeletal disease can result from analteration of a gene, wherein said alteration is

an in-frame mutation of an exon, a mutation disrupting the translationalreading frame of the gene,

a deleterious mutation that can be compensated by the inclusion of anatypical exon in the mRNA coded by said gene, and the tc-DNA iscomplementary to an ISS or TSL present in a pre-mRNA coded by said geneand facilitates inclusion of an atypical exon, or

a mutation resulting in the presence of deleterious 3′ CUGamplification(s) in a mRNA coded by said gene.

In a particular embodiment, when the alteration is an in-frame mutationof an exon, said tc-DNA can facilitate skipping of said exon. In anotherembodiment, when the alteration is a mutation disrupting thetranslational reading frame of the gene, said tc-DNA can facilitateskipping of an exon so as to restore the reading frame of the gene. Inanother embodiment, when the alteration is a mutation resulting in thepresence of deleterious 3′ CUG amplification(s) in a mRNA coded by saidgene and, said tc-DNA AON can destroy the mRNA containing saidamplification.

The tc-DNA AON presented herein are constrained DNA AON that displayimproved hybridization properties with complementary pre-mRNAs ascompared to DNA AON that employ, for example, more conventional2′-O-methyl-phosphorothioate or Morpholino chemistries. While2′-O-methyl-phosphorothioate or Morpholino DNA AON typically require 20to 24 nucleotides to achieve specific pre-mRNA target hybridization, thepresently disclosed tc-DNA AON are capable of specific pre-mRNA targethybridization with lengths of between 10 and 18 nucleotides, and morebroadly between about 6 and about 22 nucleotides, in particular between8 and 20 nucleotides.

As described in greater detail, below, exon skipping is achieved in thenucleus during the maturation process of pre-mRNAs. It includes themasking of key sequences involved in the splicing of targeted exons byusing antisense oligonucleotides (AON) that are complementary to exondefinition sequences within a pre-mRNA. Provided herein are tc-DNA AONsthat may be suitably employed for exon skipping through the masking ofsplice sites at intron/exon junctions, or more generally sites used forexon definition, within a dystrophin pre-mRNA thereby facilitating thedeletion of a deleterious exon during the processing of the pre-mRNA toa mature mRNA. Such tc-DNA AON will find utility in the treatment ofDuchenne Muscular Dystrophy by restoring an open reading frame in amutated dystrophin gene comprising an exon that contains a non-sense, astop, a frameshift mutation, or an intronic sequence that contains adeleterious cryptic exon.

For example, a non-sense or frameshift mutation within exon 23 or exon51 of a dystrophin gene yields a carboxy-terminally truncated,non-functional dystrophin protein. By hybridizing to nucleotidescomprising a dystrophin pre-mRNA splice donor site in intron 23 orintron 51, respectively, and adjacent 5′ nucleotides of exon 23 or exon51, tc-DNA AON disclosed herein are capable of preventing the inclusionof the mutated exon 23 or exon 51 into the mature mRNA transcript. Theexpression of that mature mRNA transcript yields a functional dystrophinprotein that is deleted in the amino acids encoded by exon 23 or exon 51but that includes dystrophin amino acids both N-terminal and C-terminalto those deleted amino acids and, therefore, constitutes asemi-functional ‘quasi-dystrophin’.

The tc-DNA AONs disclosed herein for skipping an exon during processingof a dystrophin pre-mRNA contain between about 6 and about 22nucleotides, in particular between about 8 and 20 tricyclo nucleotides,in particular between 10 and 18 tricyclo nucleotides, wherein 8-16nucleotides of the tc-DNA AON are complementary to a dystrophin pre-mRNAintronic splice donor site, wherein 2-8 nucleotides of the tc-DNA AONare complementary to a dystrophin pre-mRNA exonic region, and whereinthe intronic splice donor site is contiguous with and 5′ to the exonicregion. Within certain aspects, tc-DNA AONs are between 12 and 16nucleotides or between 13 and 15 nucleotides and comprise between 6 and14 nucleotides that are complementary to the intronic splice donor siteand between 2 and 5 nucleotides that are complementary to the exonicregion. It will be understood, however, that longer tc-DNA AON may besuitably employed to achieve exon skipping during processing of adystrophin pre-mRNA.

Exemplified herein are tc-DNA AONs designed for skipping a mutated exon23 within a dystrophin pre-mRNA. The tc-DNA AON comprise the nucleotidesequence 5′-AACCTCGGCTTACCT-3′ (M23D (+02−13), SEQ ID NO: 1) andspecifically hybridize to nucleotides at the 3′ end of dystrophinpre-mRNA intron 23 and to nucleotides at the contiguous 5′ end ofdystrophin pre-mRNA exon 23. Also provided are tc-DNA AON designed forskipping a mutated exon 51 within a dystrophin pre-mRNA. The tc-DNA AONcomprise a nucleotide sequence selected from the group consisting of5′-AGAAATGCCATCTTC-3′ (H51 (+68+82), SEQ ID NO: 2),5′-AAATGCCATCTTCCT-3′ (H51 (+70+84), SEQ ID NO: 3), and5′-TGCCATCTTCCTTGA-3′ (H51 (+73+87), SEQ ID NO: 4) and specificallyhybridize to nucleotides at the 3′ end of dystrophin pre-mRNA intron 51and to nucleotides at the 5′ end of dystrophin pre-mRNA exon 51.

The following nomenclature is used herein: “M” refers to mouse, “H”refers to human, “23” and “51” refer to specific exons, “D” refers todonor site, “A” refers to acceptor cite, “+” followed by a numeralindicates the number of nucleotides in the exon sequence, and “−”followed by a numeral indicates the number of nucleotides in theflanking exon. Thus, for example, M23D (+02−13) indicates that thetc-DNA AON encompasses the two 3′-terminal nucleotides of exon 23 andthe 13 5′-terminal nucleotides of intron 23, which AON is capable ofmasking the donor splice site of mouse dystrophin exon 23 and H51(+68+82) indicates that the tc-DNA AON spans from nucleotide number 68to nucleotide number 82 in human dystrophin exon 51.

Other aspects of the present disclosure provide tc-DNA AON that may besuitably employed for masking intronic silencing sequences (ISS) orterminal stem loops (TSL) within a survival motor neuron 2 (SMN2)pre-mRNA. Such tc-DNA AON facilitate the inclusion of an atypical exonduring the processing of the SMN2 pre-mRNA to a mature mRNA. Theresulting modified functional SMN2 protein contains the amino acidsequence encoded by the included atypical exon. Such a modifiedfunctional SMN2 protein is capable of complementing a non-functionalSMN1 protein and, when expressed in vivo, can at least partially reverseSpinal Muscular Atrophy that is caused by mutations in the SMN1 gene.

For example, while exon 7 of SMN2 is typically excluded from the maturemRNA transcript through processing of the corresponding pre-mRNA, theaddition of exon 7 yields a modified functional SMN2 protein that iscapable of compensating functionally for the mutated SMN1 protein. Byhybridizing to nucleotides comprising an SMN2 ISS or TSL within an SMN2pre-mRNA, a tc-DNA AON can facilitate the inclusion of exon 7 into themature mRNA transcript. The expression of that mature mRNA transcriptyields a modified functional SMN2 protein that includes the amino acidsencoded by exon 7 as well as all other SMN2 amino acids both N-terminaland C-terminal to those included amino acids.

Thus, the present disclosure provides tc-DNA AON for facilitating theinclusion of exon 7 during processing of the SMN2 pre-mRNA wherein thetc-DNA AON is 6-22 tricyclo nucleotides in length, in particular between8-20 tricyclo nucleotides, more particularly between 10-18 tricyclonucleotides in length and wherein the tc-DNA AON is complementary to anSMN2 pre-mRNA intronic silencer sequence (ISS) or a terminal stem-loop(TSL). Such tc-DNA AON may be between 13 and 17 nucleotides, between 12and 16 nucleotides, or between 13 and 15 nucleotides. Exemplified hereinare tc-DNA AON that comprise the 15-nucleotide sequence5′-CUUUCAUAAUGCUGG-3′ (SMN2i7 (10; 25), SEQ ID NO: 5), which tc-DNA AONare complementary to an SMN2 pre-mRNA ISS and which may be employed tofacilitate the inclusion of exon 7 into a processed SMN2 mRNA. Alsoexemplified herein are tc-DNA AON that comprise the 13-nucleotidesequence 5′-UUAAUUUAAGGAA-3′ (SMN2e7 (39; 51), SEQ ID NO: 6), whichtc-DNA AON are complementary to an SMN2 pre-mRNA TSL2 and which may alsobe employed to facilitate the inclusion of exon 7 into a processed SMN2mRNA. It will be understood that combinations of the tc-DNA AONpresented herein may also be employed.

Still further aspects of the present disclosure provide tc-DNA AON thatmay be suitably employed for facilitating the destruction of a mutatedDM1 mRNA. Such tc-DNA AON comprise 9-27 tricyclo nucleotides, whereinthe tc-DNA AON is complementary to a mutated DM1 mRNA comprisingdeleterious 3′ CUG amplification(s) (n>50) and wherein the tc-DNA AON iscapable of facilitating the RNase-mediated destruction of said DM1 mRNA.Tc-DNA AON may comprise between 3 and 9; between 4 and 8; or 5, 6, or 7contiguous repeats of the nucleotide sequence 5′-CAG-3′ (SEQ ID NO: 7).An exemplary tc-DNA AON for facilitating the destruction of a mutatedDM1 comprises the 15-nucleotide sequence 5′-CAGCAGCAGCAGCAG-3′ (DM1(CAG5), SEQ ID NO: 8). Another exemplary tc-DNA AON facilitating thedestruction of a mutated DM1 comprises the 21-nucleotide sequence5′-CAGCAGCAGCAGCAGCAGCAG-3′ (DM1 (CAG7), SEQ ID NO: 9).

In other aspects, the present disclosure provides methods foreliminating a mutated exon from a dystrophin mRNA, methods for includingan atypical exon within an SMN2 mRNA, and methods for destroying a DM1mRNA comprising a pathological number of 3′ CUG amplifications in acell. Each of these methods includes the step of contacting a cell witha tc-DNA AON as disclosed herein.

Within certain embodiments are provided methods for eliminating amutated exon from a dystrophin mRNA, which methods comprise the step ofcontacting a cell that expresses a dystrophin pre-mRNA with a tc-DNA AONcontaining between 6-22 tricyclo nucleotides in length, in particularbetween 8-20 tricyclo nucleotides, more particularly between 10 and 18tricyclo nucleotides, wherein 8-16 nucleotides of the tc-DNA AON arecomplementary to a dystrophin pre-mRNA intronic splice donor site,wherein 2-8 nucleotides of the tc-DNA AON are complementary to adystrophin pre-mRNA exonic region, and wherein the exonic region iscontiguous with and 3′ to the intronic splice donor site. Exemplarymethods include the step of contacting the cell with a tc-DNA AON ofbetween 12 and 16 nucleotides or between 13 and 15 nucleotides. Suitabletc-DNA AON for use in such methods comprise the nucleotide sequence5′-AACCTCGGCTTACCT-3′ (M23D (+02−13), SEQ ID NO: 1);5′-AGAAATGCCATCTTC-3′ (H51 (+68+82), SEQ ID NO: 2),5′-AAATGCCATCTTCCT-3′ (H51 (+70+84), SEQ ID NO: 3), and5′-TGCCATCTTCCTTGA-3′ (H51 (+73+87), SEQ ID NO: 4).

Within other embodiments are provided methods for including an atypicalexon within an SMN2 mRNA, which methods comprise the step of contactinga cell that is expressing an SMN2 pre-mRNA with a tc-DNA AON thatcontains between 6-22 tricyclo nucleotides in length, in particularbetween 8-20 tricyclo nucleotides, more particularly between 10 and 18or between 11 and 18 tricyclo nucleotides, wherein the tc-DNA AON iscomplementary to an SMN2 pre-mRNA intronic silencer sequence (ISS), suchas ISS-N1 within intron 7. Exemplary methods include the step ofcontacting the cell with a tc-DNA AON of between 12 and 16 nucleotidesor between 13 and 15 nucleotides. Suitable tc-DNA AON for use in suchmethods comprise the 15-nucleotide sequence 5′-CUUUCAUAAUGCUGG-3′(SMN2i7 (10; 25), SEQ ID NO: 5). Within related methods, the tc-DNA AONis complementary to an SMN2 pre-mRNA terminal stem-loop (TSL), such asTSL-2 within exon 7. Suitable tc-DNA AON for use in such methodscomprise the 13-nucleotide sequence 5′-UUAAUUUAAGGAA-3′ (SMN2e7 (39;51), SEQ ID NO: 6).

Within still further embodiments are provided methods for destroying aDM1 mRNA comprising one or more 3′ CUG amplifications in a cell, whichmethods comprise the step of contacting a cell with a tc-DNA AONcomprising 9-27 tricyclo nucleotides wherein the tc-DNA AON iscomplementary to a mutated DM1 mRNA comprising one or more 3′ CUGamplification(s) and wherein the tc-DNA AON is capable of facilitatingthe RNase-mediated destruction of the DM1 mRNA. Suitable tc-DNA AON foruse in such methods comprise between 3 and 9; between 4 and 8; or 5, 6,or 7 contiguous repeats of the nucleotide sequence 5′-CAG-3′ (SEQ ID NO:7) and are exemplified by tc-DNA AON comprising the 15-nucleotidesequence 5′-CAGCAGCAGCAGCAG-3′ (DM1 (CAG5), SEQ ID NO: 8). Anotherexemplary tc-DNA AON facilitating the destruction of a mutated DM1comprises the 21-nucleotide sequence 5′-CAGCAGCAGCAGCAGCAGCAG-3′ (DM1(CAG7), SEQ ID NO: 9).

In other aspects, the present disclosure provides methods for thetreatment of Duchenne Muscular Dystrophy (DMD), methods for thetreatment of Spinal Muscular Atrophy (SMA), and methods for thetreatment of Steinert's Myotonic Dystrophy (SD). Each of these methodsemploy the step of administering to a patient a tc-DNA AON, as disclosedherein, for eliminating a mutated exon from a dystrophin mRNA, forincluding an atypical exon within an SMN2 mRNA, or for destroying a DM1mRNA comprising one or more 3′ CUG amplifications, respectively.

These and other embodiments, features and advantages of the disclosurewill become apparent from the detailed description and the appendedclaims set forth herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table of various synthetic nucleotides used for theproduction of antisense oligonucleotides.

FIG. 2 is a structural representation of a tricyclo-DNA (tc-DNA).

FIG. 3 is a diagram showing that the mdx mouse carries a non-sensemutation in exon 23 of the dystrophin gene, which precludes synthesis offunctional dystrophin. Exon 23 partially encodes repeats R6 and R7 inwhich a C to T mutation creates a stop codon (TAA). The 15 nucleotidetc-DNA AON for exon skipping at the downstream donor splice site of exon23, designated M23D (+02−13) has the nucleotide sequence5′-AACCTCGGCTTACCT-3′ (SEQ ID NO: 1) and hybridizes to the targetdystrophin pre-mRNA exon 23/intron 23 junction, which is defined by thesequence 5′-exon 23 . . . TCAGgtaagccgaggtttggcc . . . intron 23-3′ (SEQID NO: 2), where capital letters designate exonic nucleotides andlower-case letters designate intronic nucleotides.

FIG. 4 is an agarose gel of nested RT-PCR reactions showing the skippingof dystrophin pre-mRNA exon 23 in mdx myotubes transfected, with orwithout oligofectamine, with 1, 2, and 10 μg of tc-DNA AON M23D(+02−13). After 48 hours, cultures were harvested and processed for mRNAextraction.

FIG. 5 is an agarose gel of nested RT-PCR reactions showing the skippingof dystrophin pre-mRNA exon 23 in mdx myotubes transfected with 0.5, 1,2, 5 and 10 μg of tc-DNA AON M23D (+02−13) in the presence ofoligofectamine. After 48 hours, cultures were harvested and processedfor mRNA extraction.

FIG. 6 is an agarose gel of nested RT-PCR reactions showing the skippingof dystrophin pre-mRNA exon 23 in mdx myotubes transfected with 5 μg oftc-DNA AON M23D (+02−13) in the presence of oligofectamine. Cultureswere harvested and processed for mRNA extraction from day 0 to day 15after transfection.

FIG. 7 is an agarose gel of nested RT-PCR reactions showing the skippingof dystrophin pre-mRNA exon 23 in eight week old mdx mice injected inthe tibialis anterior muscle with 50 μl PBS (phosphate buffer saline)containing 100, 80, 40, 20, 10 and 5 μg of tc-DNA AON M23D (+02−13).Animals were sacrificed 3 weeks later and muscle samples processed formRNA analysis.

FIG. 8 is an agarose gel of RT-PCR reactions showing the skipping ofdystrophin pre-mRNA exon 23 in eight week old mdx mice injected in thetibialis anterior muscle with 50 μl PBS (phosphate buffer saline)containing 10 μg of tc-DNA AON M23D (+02−13). Animals were sacrificed 4,10, and 20 weeks later and muscle samples processed for mRNA analysis.

FIG. 9 is a photomicrograph of immune-staining of dystrophin intransverse sections of tibialis anterior muscle tissue from mdx miceinjected at eight weeks with 50 μl PBS (phosphate buffer saline)containing 10 μg of tc-DNA AON M23D (+02−13). Animals were sacrificed 4,10, and 20 weeks later and muscle samples processed for immuno-staining.

FIG. 10 is a immune-staining of dystrophin in the CNS of normal and mdxmouse injected with tc-DNA AON M23D (+02−13) into the hippocampus orinto the cerebro-spinal fluid. A-B-C sections at the level of thehippocampus of normal, mdx and treated mdx with 20 μg tc-DNA M23D(+02−13), one month after a single intrathecal injection. D-E-F sectionsat the level of the cerebellum of normal, mdx and treated mdx with 200μg tc-DNA M23D (+02−13), one month after delivery in the cerebrospinalfluid. Nuclei are counterstained with DAPI.

FIG. 11 is a diagrammatic representation of the overall processing ofpre-mRNA into a mature mRNA.

FIG. 12 is an agarose gel of nested RT-PCR reactions showing theskipping of dystrophin pre-mRNA exon 51 in eight to ten-week-old hDMDmice injected in the tibialis anterior muscle with 50 μl PBS (phosphatebuffer saline) containing various tc-DNA AON.

FIG. 13 is a diagrammatic representation of the intron-exon structuresand chromosomal location of SMN1 and SMN2 genes.

FIG. 14 is a diagrammatic representation of the point mutation (C6T) inSMN2, which predominantly lacks exon 7, that affects mRNA splicing.

FIG. 15 is a diagrammatic representation of enhanced exon 7 inclusion inSMN2 by improving the use of splice acceptor (“SA”) 7 at the intron6-exon 7 boundary, and splice donor (SD) 7 at the exon 7-intron 7boundary.

FIG. 16 is a diagrammatic representation of the structure of exon 7 inSMN1 and SMN2.

FIG. 17 is a diagrammatic representation of a target sequence andputative effects of tc-DNA AON SMN2e7 (39; 51) (SEQ ID NO: 6) on SMN1and SMN2 exon 7 structure. Tc-DNA AON SMN2e7 (39; 51), with the sequence5′-UUAAUUUAAGGAAUGUG-3′, likely disrupts the structure of terminal stemloop 2 in SMN2, thereby enhancing exon 7 inclusion in SMN1 and SMN2.

FIG. 18 is a diagrammatic representation of a target sequence andputative effects of tc-DNA AON SMN2i7 (10; 25) (SEQ ID NO: 5) on SMN1and SMN2 exon 7 inclusion. Tc-DNA AON SMN2i7 (10; 25), with the sequence5′-CACUUUCAUAAUGCUGG-3′, likely prevents recognition of the intronicsilencer sequence (“ISS”)-N1, allowing for recognition of the 5′ splicesite at the exon 7-intron 7 boundary. EXINCT refers to EXtendedINhibitory ContexT. Based on extensive mutation analysis, C6U has beenshown to create an extended inhibitory context affecting exon 7definition.

FIG. 19 is an agarose gel of RT-PCR reactions (Panel B) and a normalizedplot (Panel A) showing the inclusion of exon 7 in SMN2 in fibroblastsfrom an SMA patient (G03813 cell line). After 48 hours, cultures wereharvested and processed for mRNA extraction. The plain line correspondsto mock treated control cells, the discontinued line corresponds totc-DNA AON SMN2i7 (10; 25) (referred to as tc-17) treated cells, and thedotted line corresponds to tc-DNA AON SMN2e7 (39; 51) (referred to astc-TSL) treated cells. Plots have been normalized according to the totalamount of SMN1+SMN2 (full length)+SMN2 (47) in each lane. Panel C is aWestern blot showing levels of SMN in G03813 cells transfected with theindicated tc-DNA oligonucleotides. Actin is shown as a loading control.Of note is the additive effect of tc-DNA AON SMN2i7 (10; 25) (referredto as tc-ISS7) and tc-DNA AON SMN2e7 (39; 51) (referred to as tc-TSL) onSMN2 production. Panel D is a photomicrograph showing the nuclearlocalization of SMN in tc-TSL treated cells (dark dots). Nuclei arecounterstained with DAPI.

FIG. 20 (Panel A) is a Northern blot showing decreasing levels of mutanthuman DMPK mRNAs with increasing amounts of tc-DNA AON DM1 (CAG7)(referred to as tc-DNA (CAG)₇) transfected into DM1 myoblasts in vitro.After 3 days, cultures were harvested and processed for mRNA extractionand Northern blot analysis. Panel B is a quantification plot reflectingthe ratio of mutant DMPK to normal DMPK mRNAs.

FIG. 21 (Panel A) is a Northern blot showing decreasing levels of mutanthuman DMPK mRNAs with increasing amounts of tc-DNA AON DM1 (CAG7)(referred to as tc-DNA (CAG)₇), with the sequence5′-CAGCAGCAGCAGCAGCAGCAG-3′ (SEQ ID NO: 9), injected into TA muscles ofDM1 mice expressing human DMPK mRNA with 700 CUG repeats. Panel C is aNorthern blot showing decreased levels of mutant human DMPK mRNAs when30 or 60 μg of tc-DNA AON DM1 (CAG7) was injected into TA muscles of DM1mice (n=4). Panels B and D are quantification plots reflecting the ratioof mutant human DMPK to mouse DMPK mRNAs in Northern blots from Panels Aand C, respectively.

DETAILED DESCRIPTION

The present disclosure is based upon the unexpected discovery thattricyclo-DNA (tc-DNA) antisense oligonucleotides (AON) may be suitablyemployed for masking pre-mRNA splice sites within the dystrophin gene,for masking intronic silencing sequences or terminal stem-loop sequenceswithin an SMN2 gene, or for destroying a DM1 mRNA comprising one or more3′ CUG amplifications. These discoveries will find broad application inthe treatment of genetic diseases, generally, and, more specifically, inthe treatment of Duchenne Muscular Dystrophy, Spinal Muscular Atrophy,and Steinert's Myotonic Dystrophy.

Tricyclo-DNA (tc-DNA) belongs to a new class of constrained DNA analogsthat display improved hybridizing capacities to complementary RNA. Ittiget al., Nucleic Acids Res. 32:346-353 (2004); Ittig et al., Prague,Academy of Sciences of the Czech Republic. 7:21-26 (Coll. Symp. Series,Hocec, M., 2005); Ivanova et al., Oligonucleotides 17:54-65 (2007);Renneberg et al., Nucleic Acids Res. 30:2751-2757 (2002); Renneberg etal., Chembiochem. 5:1114-1118 (2004); and Renneberg et al., JACS.124:5993-6002 (2002). Pre-mRNA/tc-DNA AON heteroduplexes disclosedherein are resistant to RNase H and, as a consequence, prevent thedestruction of the targeted pre-mRNA. The advantage of the tricyclo-DNAchemistry is that the structural properties of its backbone allow areduction in the length of an AON while retaining high affinity andhighly specific hybridization with a complementary nucleotide sequence.Unexpectedly, tc-DNA AON may be advantageously used in microgram dosagesin the in vivo setting using intramuscular application, which are atleast 10-fold less than the dosages required for conventional antisenseoligonucleotide technologies. In addition, tc-DNA retain full activitywith reduced antisense lengths. Thus, for example, tc-DNA AON of 13 to15 nucleotides are highly effective in the ex vivo and in vivoapplications exemplified by the present disclosure.

The tc-DNA AON described herein also exhibit increased in vivo stabilityas compared to existing antisense oligonucleotide chemistries such as,for example, 2′-O-methyl-phosphorothioate or Morpholino chemistries.Thus, for example, a single intramuscular injection of a tc-DNA AON ofthe present disclosure retains in vivo efficacy for more than 20 weeksfollowing administration.

Furthermore, and quite surprisingly, tc-DNA AON of the presentdisclosure, as exemplified by the tc-DNA AON designated M23D (+02−13),can be delivered into the central nervous system (CNS), either throughintra-parenchymal or intra-ventricular administration or byadministration into the subarachnoid space, to restore within theneurons of the hippocampus CA1 or within neurons of the cerebral or thecerebellar cortex, a mutated gene, such as a mutated dystrophin gene.Thus, it is demonstrated that tc-DNA AON described herein canefficiently cross the ependymal barrier.

The present disclosure will be best understood by reference to thefollowing definitions:

Definitions

As used herein, the term “tricyclo-DNA (tc-DNA)” refers to a class ofconstrained DNA analogs in which each nucleotide is modified by theintroduction of a cyclopropane ring to restrict conformationalflexibility of the backbone and to optimize the backbone geometry of thetorsion angle γ. Homobasic adenine- and thymine-containing tc-DNAs formextraordinarily stable A-T base pairs with complementary RNAs.

As used herein, the term “antisense oligonucleotide (AON)” refers to anoligonucleotide that is capable of interacting with and/or hybridizingto a pre-mRNA or an mRNA having a complementary nucleotide sequencethereby modifying gene expression. Enzyme-dependent antisenseoligonucleotides include forms that are dependent on RNase H activity todegrade target mRNA, and include single-stranded DNA, RNA, andphosphorothioate antisense. Steric blocking antisense oligonucleotides(RNase-H independent antisense) interfere with gene expression or othermRNA-dependent cellular processes by binding to a target sequence ofmRNA. Steric blocking antisense includes 2′-O alkyl antisenseoligonucleotides, Morpholino antisense oligonucleotides, andtricyclo-DNA antisense oligonucleotides. As described herein, withincertain applications tc-DNA antisense oligonucleotides may be employedin enzyme-dependent applications such as, for example, theRNase-mediated destruction of DM1 mRNA comprising one or more 3′ CUGamplifications.

As used herein, “complementary” refers to a nucleic acid molecule thatcan form hydrogen bond(s) with another nucleic acid molecule by eithertraditional Watson-Crick base pairing or other non-traditional types ofpairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) betweencomplementary nucleosides or nucleotides. In reference to the tc-DNA AONof the present disclosure, the binding free energy for a tc-DNA AON withits complementary sequence is sufficient to allow the relevant functionof the tc-DNA AON to proceed and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the tc-DNA AON tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of ex vivo orin vivo therapeutic treatment. Determination of binding free energiesfor nucleic acid molecules is well known in the art (see e.g., Turner etal., CSH Symp. Quant. Biol. LII:123-133 (1987); Frier et al., Proc. Nat.Acad. Sci. USA 83:9373-77 (1986); and Turner et al., J. Am. Chem. Soc.109:3783-3785 (1987)). Thus, “complementary” (or “specificallyhybridizable”) are terms that indicate a sufficient degree ofcomplementarity or precise pairing such that stable and specific bindingoccurs between a tc-DNA AON and a pre-mRNA or mRNA target.

It is understood in the art that a nucleic acid molecule need not be100% complementary to a target nucleic acid sequence to be specificallyhybridizable. That is, two or more nucleic acid molecules may be lessthan fully complementary. Complementarity is indicated by a percentageof contiguous residues in a nucleic acid molecule that can form hydrogenbonds with a second nucleic acid molecule. For example, if a firstnucleic acid molecule has 10 nucleotides and a second nucleic acidmolecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10nucleotides between the first and second nucleic acid moleculesrepresents 50%, 60%, 70%, 80%, 90%, and 100% complementarity,respectively. “Perfectly” or “fully” complementary nucleic acidmolecules means those in which all the contiguous residues of a firstnucleic acid molecule will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid molecule, wherein thenucleic acid molecules either both have the same number of nucleotides(i.e., have the same length) or the two molecules have differentlengths.

As used herein, the terms “precursor mRNA” or “pre-mRNA” refer to animmature single strand of messenger ribonucleic acid (mRNA) thatcontains one or more intervening sequence(s) (introns). Pre-mRNA istranscribed by an RNA polymerase from a DNA template in the cell nucleusand is comprised of alternating sequences of introns and coding regions(exons). Once a pre-mRNA has been completely processed by the splicingout of introns and joining of exons, it is referred to as “messengerRNA” or “mRNA,” which is an RNA that is comprised exclusively of exons.Eukaryotic pre-mRNAs exist only transiently before being fully processedinto mRNA. When a pre-mRNA has been properly processed to an mRNAsequence, it is exported out of the nucleus and eventually translatedinto a protein by ribosomes in the cytoplasm.

As used herein, the terms “splicing” and “processing” refers to themodification of a pre-mRNA following transcription, in which introns areremoved and exons are joined. (See, FIG. 11). Splicing occurs in aseries of reactions that are catalyzed by a large RNA-protein complexcomposed of five small nuclear ribonucleoproteins (snRNPs) referred toas a spliceosome. Within an intron, a 3′ splice site, a 5′ splice site,and a branch site are required for splicing. The RNA components ofsnRNPs interact with the intron and may be involved in catalysis.

Pre-mRNA splicing involves two sequential biochemical reactions. Bothreactions involve the spliceosomal transesterification between RNAnucleotides. In a first reaction, the 2′-OH of a specific branch-pointnucleotide within an intron, which is defined during spliceosomeassembly, performs a nucleophilic attack on the first nucleotide of theintron at the 5′ splice site forming a lariat intermediate. In a secondreaction, the 3′-OH of the released 5′ exon performs a nucleophilicattack at the last nucleotide of the intron at the 3′ splice site thusjoining the exons and releasing the intron lariat. Pre-mRNA splicing isregulated by intronic silencer sequence (ISS) and terminal stem loop(TSL) sequences.

As used herein, the terms “intronic silencer sequences (ISS)” and“terminal stem loop (TSL)” refer to sequence elements within introns andexons, respectively, that control alternative splicing by the binding oftrans-acting protein factors within a pre-mRNA thereby resulting indifferential use of splice sites. Typically, intronic silencer sequencesare between 8 and 16 nucleotides and are less conserved than the splicesites at exon-intron junctions. Terminal stem loop sequences aretypically between 12 and 24 nucleotides and form a secondary loopstructure due to the complementarity, and hence binding, within the12-24 nucleotide sequence.

As used herein, the term “Spinal Muscular Atrophy (SMA)” refers todifferent clinical types of chromosome 5-linked SMA, each having incommon a genetic cause and the manifestation of weakness due to loss ofthe motor neurons of the spinal cord and brainstem. Spinal MuscularAtrophy is caused by mutations within the survival motor neuron geneSMN1. At least one normal allele of the SMN1 gene is required for normalfunction.

The region of chromosome 5 that contains the SMN (survival motor neuron)gene has a large duplication. A large sequence that contains severalgenes occurs twice in adjacent segments. There are thus two copies ofthe gene, SMN1 and SMN2. The SMN2 gene has an additional mutation thatmakes it less efficient at making protein, though it does so in a lowlevel. SMA is caused by loss of the SMN1 gene from both chromosomes. Theseverity of SMA, ranging from SMA 1 to SMA 3, is partly related to howwell the remaining SMN 2 genes can make up for the loss of SMN 1. Oftenthere are additional copies of SMN2, and an increasing number of SMN2copies are related to less severe disease.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of this disclosure can beadministered. In one embodiment, a subject is a mammal or mammaliancell. In another embodiment, a subject is a human or human cell.

As used herein, the term “therapeutically effective amount” means anamount of tc-DNA AON that is sufficient, in the subject (e.g., human) towhich it is administered, to treat or prevent the stated disease,disorder, or condition. The tc-DNA AON of the instant disclosure,individually, or in combination or in conjunction with other drugs, canbe used to treat diseases or conditions discussed herein. For example,to treat a particular disease, disorder, or condition, the tc-DNA AONcan be administered to a patient or can be administered to otherappropriate cells evident to those skilled in the art, individually orin combination with one or more drugs, under conditions suitable fortreatment.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce an allergic or similar untoward reaction,such as gastric upset, dizziness and the like, when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

As used herein, the term “isolated” means that the referenced materialis removed from its native environment, e.g., a cell. Thus, an isolatedbiological material can be free of some or all cellular components, i.e.components of the cells in which the native material occurs naturally(e.g., cytoplasmic or membrane component).

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e. contaminants, including native materials fromwhich the material is obtained. For example, a purified tc-DNA AON ispreferably substantially free of cell or culture components, includingtissue culture components, contaminants, and the like. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof” mean±20% of the indicated range, value, or structure, unlessotherwise indicated.

As used herein, the terms “include” and “comprise” are usedsynonymously. It should be understood that the terms “a” and “an” asused herein refer to “one or more” of the enumerated components. The useof the alternative (e.g., “or”) should be understood to mean either one,both, or any combination thereof of the alternatives.

The term “about” or “approximately” means within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the term“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.

Tricyclo-DNA Antisense Oligonucleotides for the Treatment of DuchenneMuscular Dystrophy

As indicated above, within certain embodiments, the present disclosureprovides tc-DNA AON that may be suitably employed for the treatment ofDuchenne Muscular Dystrophy (DMD), a severe recessive x-linked form ofmuscular dystrophy that is characterized by rapid progression of muscledegeneration, eventually leading to loss in ambulation, paralysis, anddeath. DMD is caused by a mutation, such as a non-sense or frame-shiftmutation, within a dystrophin gene, which is located on the human Xchromosome. The dystrophin gene encodes the dystrophin protein, animportant structural component within muscle tissue which providesstructural stability to muscle fibre sarcolemma as well as to thedystroglycan complex (DGC), located at the cell membrane. A non-sense orframe-shift mutation results in premature termination of translationand, hence, a C-terminally truncated dystrophin protein.

DMD caused by one or more stop mutation(s) or frameshift mutation(s) canbe relieved by excising one or several exons so as to restore thetranslational reading frame and thereby restoring the mRNA sequencedownstream of the mutation. To achieve this, as part of the presentdisclosure, tc-DNA AON were developed to target regions within thepre-mRNA that can mask spliceosomal recognition of one or more exon(s).By targeting these regions with tc-DNA AON exons may be removed viaalternative splicing to yield mature, functional dystrophin mRNA.

Thus, the tc-DNA AON described herein are effective in facilitating theskipping of one or more mutated exons in a dystrophin gene during theprocessing of a dystrophin pre-mRNA thereby restoring the proper readingframe of the resulting dystrophin mRNA, which, when translated, yields afunctional dystrophin protein. Thus, the tc-DNA AON disclosed herein maybe used therapeutically for patients afflicted with DMD.

As used herein, the term “exon skipping” refers to the modification ofpre-mRNA splicing by the targeting of splice donor and/or acceptor siteswithin a pre-mRNA with one or more complementary antisenseoligonucleotide(s) (AONs). By blocking access of a spliceosome to one ormore splice donor or acceptor site, an AON can prevent a splicingreaction thereby causing the deletion of one or more exons from afully-processed mRNA. Exon skipping is achieved in the nucleus duringthe maturation process of pre-mRNAs. It includes the masking of keysequences involved in the splicing of targeted exons by using antisenseoligonucleotides (AON) that are complementary to splice donor sequenceswithin a pre-mRNA. The tc-DNA AON provided herein may be suitablyemployed for exon skipping through the masking of splice sites atintron/exon junctions within a dystrophin pre-mRNA thereby facilitatingthe deletion of a mutant exon during the processing of the pre-mRNA to amature mRNA.

For example, a non-sense or frameshift mutation within exon 23 or exon50 of a dystrophin gene yields a carboxy-terminally truncated,non-functional dystrophin protein. By hybridizing to nucleotidescomprising a dystrophin pre-mRNA splice donor site in intron 23 orintron 51, respectively, and adjacent 5′ nucleotides of exon 23 or exon51, tc-DNA AON disclosed herein are capable of preventing the inclusionof the mutated exon 23 or exon 51 into the mature mRNA transcript. Theexpression of that mature mRNA transcript yields a functional dystrophinprotein that is deleted in the amino acids encoded by exon 23 or exons50 and 51 but that includes dystrophin amino acids both N-terminal andC-terminal to those deleted amino acids.

The tc-DNA AON disclosed herein for skipping an exon during processingof a dystrophin pre-mRNA typically contain between 6-22 contiguoustricyclo nucleotides, in particular between 8-20 tricyclo nucleotides,more particularly between 10 and 18 contiguous tricyclo nucleotides,wherein 6-16 nucleotides, in particular 8-16 nucleotides of the tc-DNAAON are complementary to a dystrophin pre-mRNA intronic splice donorsite, wherein 2-8 nucleotides of the tc-DNA AON are complementary to adystrophin pre-mRNA exonic region, and wherein the intronic splice donorsite is contiguous with and 5′ to the exonic region. Depending upon theprecise application contemplated, tc-DNA AON may be between 12 and 16nucleotides or between 13 and 15 nucleotides and may comprise between 6and 14 nucleotides that are complementary to the intronic splice donorsite and between 2 and 5 nucleotides that are complementary to theexonic region.

Exemplified herein are tc-DNA AON designed for skipping a mutated exon23 within a dystrophin pre-mRNA. The tc-DNA AON comprise the nucleotidesequence 5′-AACCTCGGCTTACCT-3′ (M23D (+02−13), SEQ ID NO: 1) andspecifically hybridize to nucleotides at the 3′ end of dystrophinpre-mRNA intron 23 and to nucleotides at the contiguous 5′ end ofdystrophin pre-mRNA exon 23. Also provided are tc-DNA AON designed forskipping a mutated exon 51 within a dystrophin pre-mRNA. The tc-DNA AONcomprise a nucleotide sequence selected from the group consisting of5′-AGAAATGCCATCTTC-3′ (H51 (+68+82), SEQ ID NO: 2),5′-AAATGCCATCTTCCT-3′ (H51 (+70+84), SEQ ID NO: 3), and5′-TGCCATCTTCCTTGA-3′ (H51 (+73+87), SEQ ID NO: 4) and specificallyhybridize to nucleotides at the 3′ end of dystrophin pre-mRNA intron 51and to nucleotides at the 5′ end of dystrophin pre-mRNA exon 51.

Tricyclo-DNA Antisense Oligonucleotides for the Treatment of SpinalMuscular Atrophy

Within other embodiments, the present disclosure provides tc-DNA AONthat may be suitably employed for the treatment of Spinal MuscularAtrophy (SMA). SMA is caused by mutations in both copies of the SMN1gene, which in a normal cell is characterized by the presence of exons 7and 8 in fully-processed mRNA. Because normally processed SMN2 mRNA doesnot contain exons 7 or 8, the SMN2 protein cannot compensate for a lossin the functional SMN1 protein. By masking an intronic silencingsequence (ISS) and/or a terminal stem loop (TSL) within an SMN2pre-mRNA, tc-DNA AON described herein are capable of facilitating theinclusion of atypical exon 7 or exon 8 into a processed SMN2 pre-mRNA,which is translated into a modified functional SMN2 protein that iscapable of compensating for the loss of functional SMN1 protein and,when expressed in vivo, the modified functional SMN2 can at leastpartially reverse Spinal Muscular Atrophy that is caused by a mutationin the SMN1 gene.

Thus, the present disclosure provides tc-DNA AON for facilitating theinclusion of an atypical exon during processing of an SMN2 pre-mRNAwherein the tc-DNA AON is 6-22 tricyclo nucleotides in length, inparticular between 8-20 tricyclo nucleotides, more particularly between10-18 tricyclo nucleotides in length and wherein the tc-DNA AON iscomplementary to an SMN2 pre-mRNA intronic silencer sequence (ISS) or aterminal stem-loop (TSL). Such tc-DNA AON may be between 13 and 17nucleotides, between 12 and 16 nucleotides, or between 13 and 15nucleotides.

Exemplified herein are tc-DNA AON that comprise the 15-nucleotidesequence 5′-CUUUCAUAAUGCUGG-3′ (SMN2i7 (10; 25), SEQ ID NO: 5), whichtc-DNA AON are complementary to an SMN2 pre-mRNA ISS and which may beemployed to facilitate the inclusion of atypical exon 7 into a processedSMN2 mRNA. Also exemplified herein are tc-DNA AON that comprise the13-nucleotide sequence 5′-UUAAUUUAAGGAA-3′ (SMN2e7 (39; 51), SEQ ID NO:6), which tc-DNA AON are complementary to an SMN2 pre-mRNA TSL2 andwhich may also be employed to facilitate the inclusion of exon 7 into aprocessed SMN2 mRNA.

Tricyclo-DNA Antisense Oligonucleotides for the Treatment of Steinert'sMyotonic Dystrophy

Within still further embodiments, the present disclosure provides tc-DNAAON that may be suitably employed for the treatment of Steinert'sMyotonic Dystrophy that results from CUG amplifications at the 3′ end ofthe mRNA encoding DM1. It is believed that mutated DM1 mRNAs thatcontain excessive CUG amplifications are sequestered into the nucleusand accumulate to form nuclear foci. These foci are stable and arethought to bind to factors involved in the splicing machinery therebywidely affecting the transcriptome. As part of the present disclosure,it is demonstrated, by using a U7 snRNA system, that tc-DNA AON may beemployed to target the CUG sequences and facilitate the destruction ofthe mutated DM1 mRNA thereby leading to the release of the splicingfactors and removal of the nuclear foci. Without being bound to aparticular mechanistic theory, it is further believed, quitesurprisingly, that the tc-DNA AON disclosed herein are capable offacilitating destruction of mRNA containing excessive CUGamplifications.

Thus, tc-DNA AON are described that may be suitably employed forfacilitating the destruction of a mutated DM1 mRNA comprising excess CUGamplifications. Such tc-DNA AON comprise 9-27 tricyclo nucleotides,wherein the tc-DNA AON is complementary to a mutated DM1 mRNA comprisingone or more 3′ CUG amplification(s) and wherein the tc-DNA AON iscapable of facilitating the destruction of the DM1 mRNA. Depending uponthe precise application contemplated, tc-DNA AON may comprise between 3and 9; between 4 and 8; or 5, 6, or 7 contiguous repeats of thenucleotide sequence 5′-CAG-3′ (SEQ ID NO: 7). An exemplary tc-DNA AONfor facilitating the destruction of a mutated DM1 comprises the15-nucleotide sequence 5′-CAGCAGCAGCAGCAG-3′ (DM1 (CAG5), SEQ ID NO: 8).Another exemplary tc-DNA AON facilitating the destruction of a mutatedDM1 comprises the 15-nucleotide sequence 5′-CAGCAGCAGCAGCAGCAGCAG-3′(DM1 (CAG7), SEQ ID NO: 9).

Synthesis and Isolation of Tricyclo-DNA Antisense Oligonucleotides

Tc-DNA AON may be synthesized using protocols known in the art, forexample as described in Caruthers et al., Methods in Enzymol. 211:3-19(1992); Thompson et al., PCT Publication No. WO 99/54459; Wincott etal., Nucleic Acids Res. 23:2677-2684 (1995); Wincott et al., MethodsMol. Bio. 74:59 (1997); Brennan et al., Biotechnol Bioeng. 61:33-45(1998); and Brennan, U.S. Pat. No. 6,001,311.

Methodologies for the synthesis of tc-DNA and tc-DNA AON have beendescribed and are well known in the art. See, for example, Steffens andLeumann, J. Am. Chem. Soc. 121(14):3249-3255 (1999); Steffens andLeumann, J. Am. Chem. Soc. 119:11548-11549 (1997); and Wengel, U.S. Pat.No. 7,034,133. Tc-DNA may be synthesized on a commercial DNA synthesizerfrom phosphoramidites generated by conventional solid-phase cyanoethylphosphoramidite chemistry. The tc-DNA phosphoramidite building blocksmay be synthesized as described in Steffens and Leumann, C. Hely. Chim.Acta 80:2426-2439 (1997). Chain-extension cycles may be essentiallyidentical to those for natural oligodeoxynucleotide synthesis. See,Pharmacia LKB User's Manual (56-1111-56) (Gene Assembler Special/4Primers).

For example, synthesis of tc-DNA AON may be achieved by the solid-phasephosphoramidite methodology using a Pharmacia LKB Gene Assembler Specialinstrument or an Applied Biosystems PCR-MATE EP DNA Synthesizer (Model391) connected to a personal computer. Reagent solutions may be preparedaccording to the manufacturer's protocols. See, User's Manual, AppliedBiosystems PCR_MATE EP DNA Synthesizer (Model 391 (1989) and PharmaciaLKB User's Manual (56-1111-56) (Gene Assembler Special/4 Primers).1H-tetrazole (0.45 M solution in MeCN) may be obtained from Fluka.

The assembly of tricyclo-DNA AON may be performed according to thestandard synthesis cycles with the exception that a prolonged couplingtime (e.g., 6 minutes), an 11-fold excess of phosphoramidites, and theuse of a 0.07 M instead of a 0.1 M solution of tricycloadenosinebuilding block may be employed due to its poor solubility. EitherLCAA-CPG (Sigma) or polystyrene (Pharmacia) bound natural nucleosidesmay be used as starter units.

Synthesis may be performed in the trityl off mode, ending with5′-detritylated oligomers. Coupling efficiencies may be monitored byon-line trityl assay and are typically between 90 and 99%. Aftersynthesis, the solid support may be suspended in concentrated NH₃solution and left for 15 hours at 55° C. or 2 hours at room temperature.

Crude tc-DNA AON may be purified by any of a number of methodologiesknown in the art such as, for example, ultrafiltration, gelelectrophoresis, or chromatography. Ion-exchange HPLC may be achieved ona Nucleogen DEAE 60-7 (125×4 mm) column. The isolated oligonucleotidesmay be desalted over a SP-PAK C-18 cartridge (Waters) as described inSambrook et al., “Molecular Cloning: A Laboratory Manual” 11.29 (ColdSpring Harbor Laboratory Press, Plainview, N.Y., 1989). The purifiedtc-DNA AON may be dissolved in 150 mM NaCl, 10 mM Tris-HCl, pH 7.0 andincubated with alkaline phosphatase (1 mg/ml) and phosphodiesterase (2mg/ml) at 37° C. After 5 hours, the solution may be subjected to HPLCpurification.

Chemically synthesizing nucleic acid molecules with substitutions ormodifications (base, sugar or phosphate) can prevent their degradationby serum ribonucleases, which can increase their potency. See, e.g.,Eckstein et al., PCT Publication No. WO 92/07065; Perrault et al.,Nature 344:565 (1990); Pieken et al., Science 253:314 (1991); Usman andCedergren, Trends in Biochem. Sci. 17:334 (1992); Usman et al., PCTPublication No. WO 93/15187; and Rossi et al., PCT Publication No. WO91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No.6,300,074. All of the above references describe various chemicalmodifications that can be made to the base, phosphate, or sugar moietiesof the tc-DNA AON described herein.

Formulation of Tricyclo-DNA for In Vivo Administration

Tc-DNA AON described herein may be in admixture with excipients suitablefor the manufacture of aqueous suspensions. Such excipients aresuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydropropyl-methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents can be a naturally-occurring phosphatide, for example,lecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. Aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate. Dispersible powders and granulessuitable for preparation of an aqueous suspension by the addition ofwater provide the active ingredient in admixture with a dispersing orwetting agent, suspending agent and one or more preservatives.

Tc-DNA AON compositions may be in the form of a sterile injectableaqueous or oleaginous suspension. Suspensions may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents that have been mentioned above. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a non-toxic parentally acceptable diluent or solvent, forexample as a solution in 1,3-butanediol. Among the acceptable vehiclesand solvents that can be employed are water, Ringer's solution andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

The present disclosure also includes tc-DNA AON compositions preparedfor storage or administration that include a pharmaceutically effectiveamount of the desired compounds in a pharmaceutically acceptable carrieror diluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences (Mack Publishing Co., A. R. Gennaroedit., 1985). For example, preservatives and stabilizers can beprovided. These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentscan be used.

The present disclosure provides tc-DNA AON compositions and methods forfacilitating exon skipping or masking intronic silencing or terminalstem loops in a pre-mRNA or for targeting the destruction of mRNA in acell or organism. In related embodiments, this disclosure providesmethods and tc-DNA AON compositions for treating a subject, including ahuman cell, tissue or individual, having a disease or at risk ofdeveloping a disease as described herein above. In one embodiment, themethod includes administering a tc-DNA AON of this disclosure or apharmaceutical composition containing the tc-DNA AON to a cell or anorganism, such as a mammal, such that the processing of a pre-mRNA ismodified or the destruction of an mRNA is targeted. Mammalian subjectsamenable for treatment using the compositions and methods of the presentdisclosure include those suffering from one or more disorders which areamenable to such treatment such as, for example Duchenne MuscularDystrophy, Spinal Muscular Atrophy, or Steinert's Myotonic Dystrophy.

The tc-DNA AON compositions of the instant disclosure can be effectivelyemployed as pharmaceutically acceptable formulations.Pharmaceutically-acceptable formulations prevent, alter the occurrenceor severity of, or treat (alleviate one or more symptom(s) to adetectable or measurable extent) of a disease state or other adversecondition in a patient. A pharmaceutically acceptable formulationincludes salts of the above compounds, e.g., acid addition salts such assalts of hydrochloric acid, hydrobromic acid, acetic acid, and benzenesulfonic acid. A pharmaceutical composition or formulation refers to acomposition or formulation in a form suitable for administration, e.g.,systemic administration, into a cell or patient such as a human.Suitable forms, in part, depend upon the use or the route of entry, forexample transdermal or by injection. Such forms should not prevent thecomposition or formulation from reaching a target cell (i.e. a cell towhich the tc-DNA AON is desirable for delivery). For example,pharmaceutical compositions injected into the blood stream should besoluble. Other factors are known in the art, and include considerationssuch as toxicity and forms that prevent the composition or formulationfrom exerting its effect.

Pharmaceutical compositions of this disclosure can also be in the formof oil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate.

The tc-DNA AON of this disclosure may be administered to a patient byany standard means, with or without stabilizers, buffers, or the like,to form a composition suitable for treatment. When it is desired to usea liposome delivery mechanism, standard protocols for formation ofliposomes can be followed. Thus tc-DNA AON of the present disclosure maybe administered in any form, for example transdermally or by local,systemic, or intrathecal injection.

This disclosure also features the use of tc-DNA AON compositionscomprising surface-modified liposomes containing poly(ethylene glycol)lipids (PEG-modified, or long-circulating liposomes or stealthliposomes). These formulations offer a method for increasing theaccumulation of tc-DNA AON in target tissues. This class of drugcarriers resists opsonization and elimination by the mononuclearphagocytic system (MPS or RES), thereby enabling longer bloodcirculation times and enhanced tissue exposure for the encapsulatedtc-DNA AON (Lasic et al., Chem. Rev. 95:2601-2627 (1995) and Ishiwata etal., Chem. Pharm. Bull. 43:1005-1011 (1995). Long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of tc-DNA AON,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem.42:24864-24870 (1995); Choi et al., PCT Publication No. WO 96/10391;Ansell et al., PCT Publication No. WO 96/10390; Holland et al., PCTPublication No. WO 96/10392). Long-circulating liposomes are also likelyto protect tc-DNA AON from nuclease degradation to a greater extentcompared to cationic liposomes, based on their ability to avoidaccumulation in metabolically aggressive MPS tissues such as the liverand spleen.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. For example, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the tc-DNA AON of this disclosure.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular patientdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy. Following administration of tc-DNA AON compositions accordingto the formulations and methods of this disclosure, test subjects willexhibit about a 10% up to about a 99% reduction in one or more symptomsassociated with the disease or disorder being treated, as compared toplacebo-treated or other suitable control subjects.

Tc-DNA AON can be administered to cells by a variety of methods known tothose of skill in the art, including administration within formulationsthat comprise the tc-DNA AON alone, or that further comprise one or moreadditional components, such as a pharmaceutically acceptable carrier,diluent, excipient, adjuvant, emulsifier, buffer, stabilizer,preservative, or the like. In certain embodiments, the tc-DNA AON can beencapsulated in liposomes, administered by iontophoresis, orincorporated into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, bioadhesive microspheres, or proteinaceousvectors (see, e.g., PCT Publication No. WO 00/53722).

Direct injection of the tc-DNA AON of this disclosure, whethersubcutaneous, intramuscular, or intradermal, can take place usingstandard needle and syringe methodologies, or by needle-freetechnologies, such as those described in Conry et al., Clin. Cancer Res.5:2330-2337 (1999), and PCT Publication No. WO 99/31262.

Further methods for delivery of nucleic acid molecules, such as thetc-DNA AON of this disclosure, are described, for example, in Boado etal., J. Pharm. Sci. 87:1308-1315 (1998); Tyler et al., FEBS Lett.421:280-284 (1999); Pardridge et al., Proc. Nat'l Acad. Sci. USA92:5592-5596 (1995); Boado, Adv. Drug Delivery Rev. 15:73-107 (1995);Aldrian-Herrada et al., Nucleic Acids Res. 26:4910-4916 (1998); Tyler etal., Proc. Nat'l Acad. Sci. USA 96:7053-7058 (1999); Akhtar et al.,Trends Cell Bio. 2:139 (1992); “Delivery Strategies for AntisenseOligonucleotide Therapeutics,” (ed. Akhtar, 1995); Maurer et al., Mol.Membr. Biol. 16:129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol137:165-192 (1999); and Lee et al., ACS Symp. Ser. 752:184-192 (2000).These protocols can be utilized to supplement or complement delivery ofvirtually any tc-DNA AON contemplated within this disclosure.

EXAMPLES

The above disclosure generally describes the present disclosure, whichis further exemplified by the following examples. These specificexamples are described solely for purposes of illustration, and are notintended to limit the scope of this disclosure. Although specifictargets, terms, and values have been employed herein, such targets,terms, and values will likewise be understood as exemplary andnon-limiting to the scope of this disclosure.

Example 1 Use of Tricyclo-DNA Antisense Oligonucleotides to RescueDystrophin in Dystrophic Muscle Fibers

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder thatresults from mutations in the gene encoding dystrophin. Out-of-framedeletions within the dystrophin gene that encode a truncated dystrophinprotein deficiency lead to severe DMD phenotypes. Exon-skippingstrategies using tricyclo-DNA (tc-DNA) antisense oligonucleotides (AON)were developed to permit the efficient rescue of out-of-frame dystrophingene mutations thereby restoring the translational reading-frame andhence the production of functionally active dystrophin protein. Tc-DNAAON are described that, for example, hybridize to an exon 23/intron 23junction and interfere with pre-mRNA processing such that exon 23 isspliced out of the resulting processed mRNA. Alternatively, tc-DNA AONthat hybridize to an exon 51/intron 51 junction similarly interfere withpre-mRNA processing such that exon 51 is spliced out of the processedmRNA. The resulting dystrophin proteins are thus deleted in amino acidsequences encoded by exon 23 or exon 51, respectively, yet retainsufficient functionality such that the severe DMD phenotype is reversed.

Example 2 The Mdx Mouse Model

The mdx mouse is a murine model of DMD that lacks the full lengthdystrophin protein, but retains all the smaller dystrophin isoforms.Bulfield et al., Proc. Natl. Acad. Sci. USA 81:1189-1192 (1984). The mdxmouse carries a non-sense mutation in exon 23 of the dystrophin gene,which precludes functional dystrophin synthesis (see, FIG. 3). Exon 23partially encodes repeats R6 and R7 in which a C to T mutation creates astop codon (TAA).

Example 3 In Vitro Studies

This example demonstrates that mdx myotubes transfected with a15-nucleotide tc-DNA AON designated M23D (+02−13) having the nucleotidesequence 5′-AACCTCGGCTTACCT-3′ undergo exon skipping at the downstreamdonor splice site of exon 23 such that dystrophin pre-mRNA is processedto mRNA that are deleted in exon 23.

The tc-DNA AON designated M23D (+02−13), was designed such that ithybridizes to the target sequence intron 22—ttttgag[GCTC . . . EXON 23 .. . TCAG]gtaagccgaggtttggcc—intron 23 at the exon 23/intron 23 splicejunction.

Mdx myotubes were transfected with tc-DNA AON M23D (+02−13) (1, 2 and 10μg) with or without oligofectamine. One sample was left untreated, as anegative control. After 48 hours, cultures were harvested and mRNA wasextracted using the RNeasy mini kit (Qiagen). mRNA was then reversetranscribed, as follows. Eight microliters of extracted RNA (500 ng to 1μg) was mixed with 1 μL dNTP and 1 μL random hexamers, and the mixturewas incubated for 5 minutes at 65° C. The mixture was then cooled onice. 25 mM MgCl₂ (4 μL), 0.1 M DTT (2 μL), 1 μL RNase out ribonucleaseinhibitor (40 U/μL), 1X Tampon (2 μL of 10× stock) and 50 U SuperScriptreverse transcriptase were added to the mixture to bring the reaction toa final volume of 20 μL. The reaction was then incubated for 10 minutesat 25° C., followed by 50 minutes at 42° C. Next, the reaction wasinactivated by heating for 15 minutes at 70° C. The reaction was thenplaced on ice and vortexed. One μL of RNase H was then added to thereaction, and incubated at 37° C. for 20 minutes.

Next, the skipping of exon 23 was assayed by nested PCR, using thefollowing conditions. Twenty five μL PCR master mix (Taq polymerase 50U/μL, 400 μM dNTPs, 3 mM MgCl₂) was combined with 3 μL cDNA, 22 μL H₂Oand 1 μL each of the following two primers (100 μM stock concentration):

Ex 20 Fo 5′ - CAGAATTCTGCCAATTGCTGAG -3′ Ex 26 Ro 5′- TTCTTCAGCTTGTGTCATCC -3′

The reaction was then subjected to the following thermal cyclingparameters. Five min. at 94° C., followed by thirty cycles of 30 sec.94° C., 1 min. 55° C. and 2 min. at 72° C. Finally, the reaction issubjected to a five minute incubation at 72° C.

Two microliters of the PCR product was added to another PCR reaction astemplate, using 1 μL of the following primers (100 μM stockconcentration). The reaction also included 25 μL PCR master mix and 23μL H₂O.

Ex 20 Fi 5′ - CCCAGTCTACCACCCTATCAGAGC -3′ Ex 26 Ri 5′- CCTGCCTTTAAGGCTTCCTT -3′

The reaction was subjected to the following thermal cyclingparameters—Five min. at 94° C., followed by twenty five cycles of 30sec. 94° C., 1 min. 55° C. and 2 min. at 72° C. Finally, the reactionwas subjected to a five minute incubation at 72° C.

The data presented in FIG. 4 demonstrate that: (1) the 15 nucleotidetc-DNA AON M23D (+02−13) can achieve skipping of the mutated exon 23 inthe dystrophin mRNA of mdx ex vivo and (2) oligofectamine improvestc-DNA AON uptake ex vivo.

Mdx myotubes were transfected with tc-DNA AON M23D (+02−13) (0.5, 1, 2,5 and 10 μg) in the presence of oligofectamine. Cultures were processedas described above. The data presented in FIG. 5 demonstrate thatskipping is noticeable in the presence of 2 μg of tc-DNA AON M23D(+02−13).

Mdx myotubes were transfected with 5 μg of tc-DNA AON M23D (+02−13) inthe presence of oligofectamine. Cultures were processed as describedabove at different time points after transfection (from day 0 to day15). The data presented in FIG. 6 demonstrate that: (1) skipping wasobserved at day 3 (D3) and (2) skipping was still detectable at day 15(D15) but decreased from day 7 (D7).

Example 4 In Vivo Studies

This example demonstrates that mdx mice injected with a 15-nucleotidetc-DNA AON designated M23D (+02−13) having the nucleotide sequence5′-AACCTCGGCTTACCT-3′ undergo exon skipping at the downstream donorsplice site of exon 23 such that dystrophin pre-mRNA is processed tomRNA that are deleted in exon 23.

Eight-week-old mdx mice were injected in the tibialis anterior musclewith 50 μl PBS (phosphate buffer saline) containing 100, 80, 40, 20, 10and 5 μg of tc-DNA AON M23D (+02−13). Animals were sacrificed 3 weekslater. Muscle samples were processed for mRNA analysis using the sameparameters given above for example 3, and the results are given in FIG.7.

The data presented in FIG. 7 demonstrate that exon skipping occurred inall conditions tested. Also, significant levels of dystrophin proteinwere detected in transverse sections (not shown). Injection of 2 μgtc-DNA AON M23D (+02−13) was equivalent to 5 μg tc-DNA AON M23D (+02−13)(not shown).

Eight week old mdx mice were injected intramuscularly with 50 μl PBScontaining 10 μg tc-DNA AON M23D (+02−13). Animals were sacrificed at 4,10, and 20 weeks after injection. Muscle samples and transverse sectionswere assayed for dystrophin mRNA, as described above. The results arepresented in FIG. 8.

Muscle transverse sections were assayed for dystrophin proteinexpression using immunostaining, as follows. Primary monoclonal antibodyNCL-Dys 2 (1:100 dilution) was added to the samples, using the M.O.M.(mouse on mouse) kit. The samples were then washed 3 times in PBS. Thesamples were then incubated with secondary antibody goat anti-mouse IgG,labeled with Alexa Fluor 488 (Molecular Probes) for two hours, followedby a PBS wash, a 0.01% TritonX wash and a final PBS wash. The results ofthis experiment are given in FIG. 9.

The data presented in FIGS. 8 and 9 demonstrate that (1) exon skippingwas apparent at 4 and 10 weeks but was not observed at 20 weeks; (2)dystrophin is clearly detectable from 4 weeks through 20 weeks; and (3)tc-DNA AON M23D (+02−13) appears more efficient in vivo than ex vivo.

Example 5 Brain Immunostaining

Eight week old mdx mice were injected in the hippocampus or thecerebro-spinal fluid (intrathecal injection) with 50 μl PBS containing20 μg (hippocampus) or 200 μg (intrathecal) tc-DNA AON M23D (+02−13).Animals were sacrificed one month after injection. Brain sections(panels A, B, C) and cerebellar sections (panels D, E, F) were assayedfor dystrophin protein, as described above in Example 4. The results arepresented in FIG. 10. Panels A and D correspond to negative controls,where the mice were not treated. Panels B and E correspond to untreatedsection from an mdx mouse. Panel C corresponds to a hippocampus-treatedmdx mouse (C), and panel (F) shows the results from anintrathecal-treated cerebellum of mdx mouse. Nuclei were counterstainedwith DAPI.

Example 6 Delivery (Prophetic)

Tc-DNA AON M23D (+01−13) may be delivered through intraperitoneal andsubcutaneous injections (from 1 g/kg and below).

Example 7 Tricyclo-DNA Antisense Oligonucleotides for Dystrophin Rescuein DMD Mice

This example demonstrates that tc-DNA AON designed for the dystrophinexon 51/intron 51 junction having the sequences 5′-AGAAATGCCATCTTC-3′(“tc-DNA AON H51 (+68+82)”; SEQ ID NO: 2), 5′-AAATGCCATCTTCCT-3′(“tc-DNA AON H51 (+70+84)”; SEQ ID NO: 3), and 5′-TGCCATCTTCCTTGA-3′(“tc-DNA AON H51 (+73+87)”; SEQ ID NO: 4), effectively mediated theskipping of exon 51 in muscle cells from mice expressing the full humandystrophin gene (“hDMD mice”).

Eight to ten-week-old hDMD mice were injected in the tibialis anteriormuscle with 50 μl PBS containing 10 μg of tc-DNA AON H51 (+68+82) alsoreferred to as Tc-DNA ex51 ESEa, tc-DNA AON H51 (+70+84) also referredto as Tc-DNA ex51 ESEb, or tc-DNA AON H51 (+73+87) also referred to asTc-DNA ex51 ESEc. Animals were sacrificed 4 weeks later. Muscle sampleswere processed for mRNA analysis using the same parameters given abovefor example 3, and the results are given in FIG. 12.

The skipping of exon 51 was assayed by nested PCR under the followingconditions. Five-hundred nanograms of total RNA were used for RT-PCRusing the Access RT-PCR System (Promega) in a 50 μL reaction with thefollowing external primers:

Hex 49F2 (5′-AAACTGAAATAGCAGTTCAAGC-3′)Hex 53R2 (5′-TTGCCTCCGGTTCTGAAGG-3′)

The cDNA synthesis was carried out at 45° C. for 45 min, directlyfollowed by primary PCR for 20 cycles with the following parameters:twenty cycles of 40 sec. at 94° C., 40 sec. at 60° C., and 40 sec at 72°C.

Two microliters of the PCR product were added to another PCR reaction astemplate, using the following primers (100 μM stock concentration).

Hex 50F 5′-AGGAAGTTAGAAGATCTGAGC-3′ Hex 52R2 5′-TTCTTCCAACTGGGGACGC-3′

The reaction was subjected to the following thermal cycling parameters:Five min. at 94° C., followed by thirty cycles of 40 sec. at 94° C., 40sec. at 60° C., and 40 sec at 72° C. Finally, the reaction was subjectedto a five minute incubation at 72° C.

The data presented in FIG. 12 demonstrate that each tc-DNA AON H51construct tested (tc-DNA AON H51 (+68+82), tc-DNA AON H51 (+70+84), andtc-DNA AON H51 (+73+87)) resulted in increased exon 51 skipping.

Example 8 Tricyclo-DNA Antisense Oligonucleotides Directed to the SMNExon 7/Intron 7 Junction and Intron 7 ISS Promote Exon 7 Inclusion inSMN2

This example demonstrates that tc-DNA AON designed for the SMN exon7/intron 7 junction and intron 7 ISS (“tc-DNA AON SMN2e7 (39; 51)” and“tc-DNA AON SMN2i7 (10; 25)”, respectively) effectively mediates theinclusion of exon 7 in SMN2 in fibroblast cells (G03813 cell line)isolated from an SMA patient.

(SEQ ID NO: 5) tc-DNA AON SMN2i7(10; 25): 5′-CUUUCAUAAUGCUGG-3′(SEQ ID NO: 6) tc-DNA AON SMN2e7(39; 51): 5′-UUAAUUUAAGGAA-3′

The G03813 cell line originates from a 3-year-old type I SMA patientwith two copies of SMN2. GM03813 cells were cultured in Dulbecco'sModified Eagle Medium (DMEM) with 20% fetal bovine serum and 1%penicillin-streptomycin (100 U/ml). tcDNA AON (tc-DNA AON SMN2i7 (10;25) and tc-DNA AON SMN2e7 (39; 51)) transfections were carried out withOligofectamine (Invitrogen) in medium without serum and antibiotics for48 hours. Total RNA was extracted 48 hours post transfection usingTRIzol reagent (Invitrogen) and first-strand cDNA synthesis wasperformed using SuperScript II (Invitrogen) and random hexamers. PCR wascarried out using Master Mix 2X Phusion GC (Finnzymes) in a total volumeof 50 μL with 11 μL of cDNA and 10 μM each of SMN-Ex6-FW and SMN-Ex8-Reprimers. PCR products were then separated by electrophoresis on a 3%agarose gel.

SMN-Ex6-Fw 5′-GCTGATGCTTTGGGAAGTATGTA-3′SMN-Ex8-Re 5′-ATTCCAGATCTGTCTGATCG-3′

The data presented in FIG. 19 show that both tc-DNA AON SMN2e7 (39; 51)and tc-DNA AON SMN2i7 (10; 25) promote exon 7 inclusion in SMN2 mRNA.Panel A shows RT-PCR analysis of RNA from GM03813 cells treated with theindicated tc-DNA oligonucleotides. The intensity of the bandcorresponding to SMN2 without exon 7 (“SMN2 (Δ7)”) is lower with bothtc-DNA oligonucleotides (lanes 3 and 4) compared to the band in theuntreated lane (lane 2). Panel B is a normalized quantification plot ofthe gel showing that upon tc-DNA AON transfection, the upper bandcorresponding to SMN1+full length SMN2 increased, while the lower bandcorresponding to SMN2 (Δ7) significantly decreased.

Panel C shows a Western blot of lysates from wild type fibroblasts andGM03813 cells transfected with the indicated tc-DNA oligonucleotides.Protein extracts were obtained in lysis buffer (10 mmol/l HEPES pH 7.9,100 mmol/l KCl, 1 mmol/l EDTA, 1 mmol/l 1,4-dithiothreitol, 1× completeprotease inhibitor cocktail (Roche), 0.5% NP-40). Equal amounts ofprotein (determined by Bradford Protein Assay (Pierce)) were mixed with2× loading buffer (125 mmol/l Tris pH 6.8, 2% sodium dodecyl sulfate,10% glycerol, 0.01% bromophenol blue, 10% β-mercaptoethanol) and proteinconcentration was determined using the Bradford Protein Assay (Pierce).Ten micrograms of each protein sample were resolved by SDS-PAGE 4-12%Bis-Tris Gels (Invitrogen) and transferred onto a nitrocellulosemembrane. The membrane was blocked with 10% milk in PBS-Tween buffer,probed with a rabbit polyclonal SMN antibody (dilution 1:500; h-195,Santa Cruz) which recognizes SMN1, SMN2, and the truncated form of SMN2,and then incubated with a goat anti-rabbit secondary antibody conjugatedwith horseradish peroxidase (1:50,000). Signals were detected with theSuperSignal West Pico Chemiluminescent kit (ThermoScientific). Toconfirm equal loading of proteins, the membrane was washed, reblocked,and probed with a mouse monoclonal anti-actin antibody, followed byincubation with a secondary sheep anti-mouse antibody conjugated withhorseradish peroxidase (1:15,000). Signals were detected as describedabove. Lane 1: GM03813 cells transfected with tc-DNA AON SMN2i7 (10;25); Lane 2: GM03813 cells transfected with tc-DNA AON SMN2e7 (39; 51);Lane 3: GM03813 cells transfected with both tc-DNA AON SMN2i7 (10; 25)and tc-DNA AON SMN2e7 (39; 51); Lane 4: wild type fibroblasts; Lane 5:Non-transfected GM03813 cells.

The data in panel C indicate transfection with tc-DNA oligonucleotidesrescued SMN levels in GM03813 cells due to inclusion of exon 7 in SMN2mRNA. Control GM03813 cells display some SMN protein due to sporadicnatural inclusion of exon 7 in SMN2.

Panel D shows a photomicrograph of GM03813 cells transfected with 30 μgtc-TSL and subsequently stained with SMN antibody. Transfected GM03813cells on slides were fixed with acetone/methanol (volume/volume). Fixedcells were blocked in PBS+5% BSA for 1 hour, followed by incubation witha rabbit polyclonal SMN antibody (1:100 in PBS+1% BSA; h-195, SantaCruz) for 1 hour. Cells were washed in PBS and incubated with asecondary anti-rabbit antibody conjugated to Alexa 594 for 1 hour. Cellswere then washed in PBS and incubated with DAPI (1:50,000) for 5minutes. Slides were fitted with coverslips using Fluoromount-G(SouthernBiotech) and incubated overnight at 4° C. The photomicrographshows that SMN (red) levels increased in the nuclei (blue) of GM03813cells transfected with tc-DNA TSL.

Example 9 Tricyclo-DNA Antisense Oligonucleotides Directed to CUGRepeats Reduce Mutant DMPK mRNA Expression in DM1 Myoblasts

This example demonstrates that tc-DNA AON designed for CUG repeats inmutant DMPK mRNA having the sequence 5′-CAGCAGCAGCAGCAGCAGCAG′3′(“tc-DNA AON DM1 (CAG7)”; SEQ ID NO: 9) effectively reduces theexpression of mutant DMPK mRNA with 800 CUG repeats in human DM1myoblasts isolated from muscle biopsy obtained from the Tissue Bank forResearch “Myobank”.

DM1 myoblasts were transfected with increasing amounts of tc-DNA AON DM1(CAG7) (0, 3.5 μg, 10 μg, and 20 μg) using Lipofectamine (Invitrogen).Three days after transfection, the expression of wild type and mutantDMPK mRNAs were detected by Northern blot. Briefly, 5-10 μg of total RNAwere separated on 1.3% agarose MOPS-gels containing 0.66 M formaldehydeand transferred onto Hybond-N+ membranes (Amersham Pharmacia Biotech) bycapillary transfer with 10×SSC. Blots were hybridized with arandom-primed ³²P-labeled (Bgl II-Sac I fragment of DMPK cDNA) probe inhybridization buffer (2% SDS, 10% dextran sulfate, 1×SSPE, 100 μg/mlsalmon sperm DNA, 2% Denhart's) at 68° C. overnight. Signals wereanalyzed on a phospho-imager (Molecular Imager FX, Bio-Rad) andquantified using Quantity One (Bio-Rad).

Panel A of FIG. 20 shows that levels of mutant DMPK decreased withincreasing amounts of transfected tc-DNA AON DM1 (CAG7). Importantly,levels of wild type DMPK were unaltered. 18S RNA was used as a loadingcontrol. Panel B is a quantification plot of the Northern blot fromPanel A. Quantification was performed by measuring the band intensityratio of mutant DMPK to wild type DMPK.

Example 10 Tc-DNAs Directed to CUG Repeats Reduce Mutant DMPK mRNAExpression in DM1 Mice

This example demonstrates that tc-DNA AON DM1 (CAG7) effectively reduceslevels of mutant human DMPK mRNA with 700 CUG repeats expressed in TAmuscles of DM1 mice.

TA muscles of DM1 mice expressing human DMPK mRNA with 700 CUG repeatswere injected with increasing amounts of tc-DNA AON DM1 (CAG7). One weeklater, total RNA was extracted using TRIzol reagent (Invitrogen). HumanDMPK and mouse DMPK mRNAs were detected by Northern blot. Briefly, 8-10μg of total RNA were separated on 1.3% agarose MOPS-gels containing 0.66M formaldehyde and transferred onto Hybond-N+ membranes (AmershamPharmacia Biotech) by capillary transfer with 10×SSC. Blots werehybridized with a random-primed ³²P-labeled (Bgl II-Sac I fragment ofDMPK cDNA) probe in hybridization buffer (2% SDS, 10% dextran sulfate,1×SSPE, 100 μg/ml salmon sperm DNA, 2% Denhart's) at 68° C. overnight.Signals were analyzed on a phospho-imager (Molecular Imager FX, Bio-Rad)and quantified using Quantity One (Bio-Rad).

Panels A and C of FIG. 21 show that levels of mutant human DMPKdecreased with increasing amounts of transfected tc-DNA AON DM1 (CAG7).Panels B and D are quantification plots of Northern blots from Panels Aand C, respectively. Quantification was performed by measuring the bandintensity ratio of mutant human DMPK to wild type mouse DMPK.

While the disclosure has been described in each of its variousembodiments, it is expected that certain modifications thereto may beundertaken and effected by the person skilled in the art withoutdeparting from the true spirit and scope of the disclosure, as set forthin the previous description and as further embodied in the followingclaims. The present disclosure is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe disclosure in addition to those described herein will becomeapparent to those skilled in the art from the foregoing description andthe accompanying figures. Such modifications are intended to fall withinthe scope of the appended claims. It is further to be understood thatall values are approximate, and are provided for description.

All U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications, non-patentpublications, figures, tables, and websites referred to in thisspecification are expressly incorporated herein by reference, in theirentirety.

SEQUENCE LISTING

SEQUENCE LISTING SEQ ID NO: 1 Artificial Sequence DNA Length: 15tc-DNA AON M23D (+02 −13) aacctcggcttacct SEQ ID NO: 2Artificial Sequence DNA Length: 15 tc-DNA AON H51 (+68 +82)agaaatgccatcttc SEQ ID NO: 3 Artificial Sequence DNA Length: 15tc-DNA AON H51 (+70 +84) aaatgccatcttcct SEQ ID NO: 4Artificial Sequence DNA Length: 15 tc-DNA AON H51 (+73 +87)tgccatcttccttga SEQ ID NO: 5 Artificial Sequence DNA Length: 15tc-DNA AON SMN2i7 (10; 25) cuuucauaaugcugg SEQ ID NO: 6Artificial Sequence DNA Length: 13 tc-DNA AON SMN2e7 (39; 51)uuaauuuaaggaa SEQ ID NO: 7 Artificial Sequence DNA Length: 12-21tc-DNA AON DM1(CAG_(n)) (cag)_(n) n = 4-7 SEQ ID NO: 8Artificial Sequence DNA Length: 15 tc-DNA AON DM1(CAG5) cagcagcagcagcagSEQ ID NO: 9 Artificial Sequence DNA Length: 21 tc-DNA AON DM1(CAG7)cagcagcagcagcagcagcag

1. A tricyclo-DNA antisense oligonucleotide (tc-DNA AON) forfacilitating the skipping of an exon during processing of a dystrophinpre-mRNA, said tc-DNA AON containing between 10 and 18 tricyclonucleotides, wherein 8-16 or 6-14 nucleotides of said tc-DNA AON arecomplementary to a dystrophin pre-mRNA intronic splice donor site,wherein 2-8 nucleotides of said tc-DNA AON are complementary to adystrophin pre-mRNA exonic region, and wherein said intronic splicedonor site is contiguous with and 5′ to said exonic region. 2.-4.(canceled)
 5. The tc-DNA AON of claim 1 wherein the exon that is skippedduring processing of said dystrophin pre-mRNA is exon 23 or exon
 51. 6.The tc-DNA AON of claim 5 wherein, when the skipped exon is exon 23,said tc-DNA AON comprises the nucleotide sequence 5′-AACCTCGGCTTACCT-3′(SEQ ID NO: 1) or is M23D (+02−13) (SEQ ID NO: 1), and, when the skippedexon is exon 51, said tc-DNA AON comprises a nucleotide sequenceselected from the group consisting of 5′-AGAAATGCC ATCTTC-3′ (SEQ ID NO:2), 5′-AAATGCCATCTTCCT-3′ (SEQ ID NO: 3), and 5′-TGCCATCTTCCTTGA-3′ (SEQID NO: 4) or is H51 (+68+82) (SEQ ID NO: 2) or is H51 (+70+84) (SEQ IDNO: 3) is H51 (+73+87) (SEQ ID NO: 4).
 7. A tricyclo-DNA antisenseoligonucleotide (tc-DNA AON) for facilitating the inclusion of anatypical exon during processing of an SMN2 pre-mRNA, said tc-DNA AONcontaining between 10 and 18 tricyclo nucleotides, wherein said tc-DNAAON is complementary to an SMN2 pre-mRNA intronic silencer sequence(ISS).
 8. (canceled)
 9. The tc-DNA AON of claim 7 wherein said atypicalexon in said SMN2 pre-mRNA is exon 7 and said intronic silencer sequenceis ISS-N1.
 10. The tc-DNA AON of claim 9 wherein said tc-DNA AONcomprises the nucleotide sequence 5′-CUUUCAUAAUGCUGG-S′ (SEQ ID NO: 5)or is SMN2i7 (10; 25) (SEQ ID NO: 5).
 11. A tricyclo-DNA antisenseoligonucleotide (tc-DNA AON) for facilitating the inclusion of anatypical exon during processing of an SMN2 pre-mRNA, said tc-DNA AONconsisting of 10-18 tricyclo nucleotides, wherein said tc-DNA AON iscomplementary to an SMN2 pre-mRNA terminal stem-loop (TSL). 12.(canceled)
 13. The tc-DNA AON of claim 11 wherein said atypical exon insaid SMN2 pre-mRNA is exon 7 and said terminal stem-loop is TSL2. 14.The tc-DNA AON of claim 13 wherein said tc-DNA AON comprises thenucleotide sequence 5′-UUAAUUUAAGGAA-3′ (SEQ ID NO: 6) or is SMN2e7 (39;51) (SEQ ID NO: 6).
 15. A composition for facilitating the skipping ofan exon during processing of a dystrophin pre-mRNA, for facilitating theinclusion of an atypical exon during processing of an SMN2 pre-mRNA, orfor facilitating the inclusion of an atypical exon during processing ofan SMN2 pre-mRNA, said composition comprising: (a) when the compositionis for facilitating the skipping of an exon during processing of adystrophin pre-mRNA, a tricyclo-DNA antisense oligonucleotide (tc-DNAAON) containing between 10 and 18 tricyclo nucleotides, wherein 8-16nucleotides of said tc-DNA AON are complementary to a dystrophinpre-mRNA intronic splice donor site, wherein 2-8 nucleotides of saidtc-DNA AON are complementary to a dystrophin pre-mRNA exonic region, andwherein said exonic region is contiguous with and 3′ to said intronicsplice donor site; or when the composition is for facilitating theinclusion of an atypical exon during processing of an SMN2 pre-mRNA, atricyclo-DNA antisense oligonucleotide (tc-DNA AON) containing between10 and 18 tricyclo nucleotides, wherein said tc-DNA AON is complementaryto an SMN2 pre-mRNA intronic silencer sequence (ISS); or when thecomposition is for facilitating the inclusion of an atypical exon duringprocessing of an SMN2 pre-mRNA, a tricyclo-DNA antisense oligonucleotide(tc-DNA AON) containing between 10 and 18 tricyclo nucleotides, whereinsaid tc-DNA AON is complementary to an SMN2 pre-mRNA terminal stem-loop(TSL); and (b) a cell delivery agent. 16.-17. (canceled)
 18. A methodfor eliminating a mutated exon from a dystrophin mRNA, said methodcomprising the step of contacting a cell that expresses a dystrophinpre-mRNA with a tricyclo-DNA antisense oligonucleotide (tc-DNA AON),said tc-DNA AON containing between 10 and 18 tricyclo nucleotides,wherein 8-16 nucleotides of said tc-DNA AON are complementary to adystrophin pre-mRNA intronic splice donor site, wherein 2-8 nucleotidesof said tc-DNA AON are complementary to a dystrophin pre-mRNA exonicregion, and wherein said exonic region is contiguous with and 3′ to saidintronic splice donor site.
 19. (canceled)
 20. The method of claim 18wherein the exon that is skipped during processing of said dystrophinpre-mRNA is exon 23 or
 51. 21. The method of claim 20 wherein when theskipped exon is 23, said tc-DNA AON comprises the nucleotide sequence5′-AACCTCGGCTTACCT-3′ (SEQ ID NO: 1) or is M23D (+02−13) (SEQ ID NO: 1),and when the skipped exon is exon 51, said tc-DNA AON comprises anucleotide sequence selected from the group consisting of5′-AGAAATGCCATCTTC-3′ (SEQ ID NO: 2), 5′-AAATGCCATCTTCCT-3′ (SEQ ID NO:3), and 5′-TGCCATCTTCCTTGA-3′ (SEQ ID NO: 4) or is H51 (+68+82) (SEQ IDNO: 2) or is H51 (+70+84) (SEQ ID NO: 3) or is H51 (+73+87) (SEQ ID NO:4). 22.-23. (canceled)
 24. A method for including an atypical exonwithin an SMN2 mRNA, said method comprising the step of contacting acell that is expressing an SMN2 pre-mRNA with a tc-DNA AON that containsbetween 11 and 18 nucleotides wherein said tc-DNA AON is complementaryto an SMN2 pre-mRNA intronic silencer sequence (ISS) or is complementaryto an SMN2 pre-mRNA terminal stem-loop (TSL).
 25. (canceled)
 26. Themethod of claim 24 wherein said atypical exon in said SMN2 pre-mRNA isexon 7 and said intronic silencer sequence is ISS-N1 or said terminalstem-loop is TSL2.
 27. (canceled)
 28. The method of claim 26 whereinsaid tc-DNA AON is complementary to an ISS, said tc-DNA AON comprisesthe nucleotide sequence 5′-CUUUCAUAAUGCUGG-S′ (SEQ ID NO: 5) or isSMN2i7 (10; 25) (SEQ ID NO: 5) and when said tc-DNA AON is complementaryto a TSL, said tc-DNA AON comprises the nucleotide sequence5′-UUAAUUUAAGGAA-3′ (SEQ ID NO: 6) or is SMN2e7 (39; 51) (SEQ ID NO: 6).29.-33. (canceled)
 34. A method for the treatment of Duchene MuscularDystrophy (DMD) in a patient, said method comprising the step ofadministering to said patient a tricyclo-DNA (tc-DNA) antisenseoligonucleotide (AON); wherein said tc-DNA AON comprises a nucleotidesequence that is complementary to a dystrophin pre-mRNA intron-exonjunction; wherein said intron-exon junction comprises an intronic splicedonor site that is 5′ to an exon; wherein said exon comprises a nonsenseor a frameshift mutation as compared to an exon having a wild-typenucleotide sequence; wherein said tc-DNA AON facilitates the skipping ofsaid exon during the processing of said dystrophin pre-mRNA to a maturemRNA.
 35. The method of claim 34 wherein: said tc-DNA AON has a lengthselected from the group consisting of: (i) between 11 and 18 nucleotidesor (ii) 15 nucleotides; or- said tricyclo-DNA oligonucleotide comprisesbetween 6 and 10 nucleotides that are complementary to a splice donorsite within an intron; or said tricyclo-DNA oligonucleotide comprisesbetween 6 and 10 nucleotides that are complementary to 5′ nucleotideswithin said exon; or said mutation in said exon is a non-sense mutationor a frame-shift mutation. 36.-38. (canceled)
 39. A method for thetreatment of Duchenne Muscular Dystrophy in a patient, said methodcomprising the step of administering to said patient a tricyclo-DNAoligonucleotide; wherein said tricyclo-DNA oligonucleotide comprises asequence of nucleotides that is complementary to an intron-exon junctionwithin a dystrophin pre-mRNA, wherein said intron-exon junctioncomprises a splice donor site within intron 51 and 5′ nucleotides withinadjacent exon 51 of said dystrophin pre-mRNA, wherein said dystrophinpre-mRNA comprises a mutation in said exon 51, and wherein saidtricyclo-DNA oligonucleotide is capable of mediating the skipping ofsaid exon
 51. 40. The method of claim 39 wherein; said intron-exonjunction comprises the sequence of SEQ ID NO: 1 ; or said mutation insaid exon 51 is a non-sense mutation or a frame-shift mutation; or saidtricyclo-DNA oligonucleotide comprises the sequence of SEQ ID NO: 2.41.-49. (canceled)
 50. A method for destroying a DM1 mRNA comprising oneor more 3′ CUG amplifications in a cell, said method comprising the stepof contacting said cell with a tc-DNA AON comprising 12-21 tricyclonucleotides wherein said tc-DNA AON is complementary to a mutated DM1mRNA comprising one or more 3′ CUG amplification(s) and wherein saidtc-DNA AON is capable of facilitating the RNAse H-mediated destructionof said DM1 mRNA.
 51. A method for the treatment of Steinert's MyotonicDystrophy in a patient, said method comprising the step of administeringto said patient a tc-DNA AON comprising 12-21 tricyclo nucleotideswherein said tc-DNA AON is complementary to a mutated DM1 mRNAcomprising one or more 3′ CUG amplification(s) and wherein said tc-DNAAON is capable of facilitating the RNAse H-mediated destruction of saidDM1 mRNA.
 52. A method for correcting abnormal gene expression in a cellof the central nervous system of a subject, the method comprisingadministering to the subject a tc-DNA antisense oligonucleotide, whereinsaid tc-DNA antisense oligonucleotide is complementary to a portion ofan RNA encoded by said gene, and wherein said tc-DNA antisenseoligonucleotide is administered peripherally to the subject in an amountsufficient to correct said abnormal expression.
 53. A method fortreating a genetic disease caused by abnormal gene expression in thecentral nervous system of a subject, the method comprising administeringto the subject a tc-DNA antisense oligonucleotide, wherein said tc-DNAantisense oligonucleotide is complementary to a portion of an RNAencoded by said gene, and wherein said tc-DNA antisense oligonucleotideis administered peripherally to the subject in an amount effective tocorrect said abnormal expression.
 54. A pharmaceutical compositioncomprising a tc-DNA antisense oligonucleotide wherein said tc-DNAantisense oligonucleotide is complementary to a portion of an RNAencoded by a human gene, and wherein said composition further comprisesa pharmaceutically acceptable excipient.
 55. (canceled)
 56. A methodaccording to claim 52 wherein said abnormal gene expression leads to aneuromuscular or musculoskeletal disorder.
 57. A method according toclaim 53 wherein said disease is a neuromuscular or musculoskeletaldisorder. 58.-63. (canceled)
 64. The method according to claim 52,wherein said abnormal gene expression results from an in-frame mutationof an exon, a mutation disrupting the translational reading frame of thegene, and the tc-DNA facilitates skipping of an exon so as to restorethe reading frame; a deleterious mutation that can be compensated by theinclusion of an atypical exon in the mRNA coded by said gene, and thetc-DNA is complementary to an ISS or TSL present in a pre-mRNA coded bysaid gene and facilitates inclusion of an atypical exon, or a mutationresulting in the presence of deleterious 3′ CUG amplification(s) in amRNA coded by said gene.
 65. The method according to claim 52, whereinsaid tc-DNA antisense oligonucleotide is as defined in claim
 1. 66.-69.(canceled)
 70. The method according to any one of claims 53, whereinsaid abnormal gene expression results from an in-frame mutation of anexon, a mutation disrupting the translational reading frame of the gene,and the tc-DNA facilitates skipping of an exon so as to restore thereading frame; a deleterious mutation that can be compensated by theinclusion of an atypical exon in the mRNA coded by said gene, and thetc-DNA is complementary to an ISS or TSL present in a pre-mRNA coded bysaid gene and facilitates inclusion of an atypical exon, or a mutationresulting in the presence of deleterious 3′ CUG amplification(s) in amRNA coded by said gene.
 71. The method according to any one of claims54, wherein said abnormal gene expression results from an in-framemutation of an exon, a mutation disrupting the translational readingframe of the gene, and the tc-DNA facilitates skipping of an exon so asto restore the reading frame; a deleterious mutation that can becompensated by the inclusion of an atypical exon in the mRNA coded bysaid gene, and the tc-DNA is complementary to an ISS or TSL present in apre-mRNA coded by said gene and facilitates inclusion of an atypicalexon, or a mutation resulting in the presence of deleterious 3′ CUGamplification(s) in a mRNA coded by said gene.